Chemiluminescent 3-(substituted adamant-2&#39;-ylidene) 1,2-dioxetanes

ABSTRACT

Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by the formula: ##STR1## wherein: X and X 1  each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X 1  being other than hydrogen; and 
     R 1  and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound&#39;s 4-carbon atom, with the provisos that if R 1  and R 2  represent individual substituents the R 2  substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of R 1  and R 2  is, or R 1  and R 2  taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that produces a luminescent substance when the enzymatically cleavable labile substituent thereof is removed by an enzyme. 
     The corresponding dioxetanes which, instead of being substituted at the 5&#39; or 7&#39;, or at the 5&#39; and 7&#39; positions, instead contain a 4&#39; methylene group, are also disclosed, as are intermediates for all these 3-substituted adamant-2&#39;-ylidenedioxetanes, and their use as reporter molecules in assays.

This application is a continuation of U.S. patent application Ser. No.08/233,085, filed Apr. 25, 1994, now U.S. Pat. No. 5,543,295, which adivisional of U.S. patent application Ser. No. 07/806,928, filed Dec.12, 1991, now U.S. Pat. No. 5,330,900 which is a divisional of U.S.patent application Ser. No. 07/574,786, filed Aug. 30, 1990, now U.S.Pat. No. 5,112,960.

FIELD OF THE INVENTION

This invention relates to improved chemiluminescent 1,2-dioxetanecompounds. More particularly, this invention relates to improvedenzymatically cleavable chemiluminescent 1,2-dioxetane compounds thatcontain enzymatically removable labile groups. Such labile groupsprevent the molecule from decomposing to produce light, i.e., visiblelight or light detectable by appropriate instrumentation, until anappropriate enzyme is added to remove the labile group.

One enzyme molecule will effect the removal, through a catalytic cycle,of its complementary labile group from thousands of enzymaticallycleavable chemiluminescent 1,2-dioxetane molecules. This is in markedcontrast to the situation with chemically cleavable chemiluminescent1,2-dioxetanes, where one molecule of chemical cleaving agent is neededto remove the complementary labile group from each dioxetane molecule.For example, one mole of sodium hydroxide is needed to cleave one moleof hydrogen ions from the hydroxyl substituent on the phenyl group in3-(2'-spiro-adamantane)-4-methoxy-4-(3"-hydroxy)phenyl-1,2-dioxetane,while only a single mole of alkaline phosphatase ("AP") is needed tocleave the phosphoryloxy group in 1,000-5,000 moles of3-(2'-spiroadamantane)-4-methoxy-4-(3"phosphoryloxy)phenyl-1,2-dioxetane disodium salt per second; seeJablonski, "DNA Probes for Infectious Diseases" (Boca Raton, Fla.:CRCPress, 1989), p. 22.

Enzymatically cleavable light-producing 1,2-dioxetane compounds willusually also contain stabilizing groups, such as an adamantylidene groupspiro bonded to the dioxetane ring's 3-carbon atom, that will aid inpreventing the dioxetane compound from undergoing substantialdecomposition at room temperature (about 25° C.) before the bond bywhich the enzymatically cleavable labile group is attached to theremainder of the molecule is intentionally cleaved, a concept introducedto 1,2-dioxetane chemistry by Wierynga, et al., Tetrahedron Letters, 169(1972) and McCapra, et al., J.Chem.Soc., Chem.Comm., 944 (1977). Thesestabilizing groups thus permit such dioxetanes to be stored foracceptably long periods of time before use, e.g., for from about 12months to as much as about 12 years at temperatures ranging from about4° to about as much as 30° C., without undergoing substantialdecomposition.

This invention further relates to the incorporation of its dioxetanemolecules in art-recognized immunoassays, chemical assays and nucleicacid probe assays, and to their use as direct chemical/physical probesfor studying the molecular structures or microstructures of variousmacromolecules, synthetic polymers, proteins, nucleic acids, catalyticantibodies, and the like, to permit an analyte--the chemical orbiological substance whose presence, amount or structure is beingdetermined--to be identified or quantified.

BACKGROUND OF THE INVENTION

Chemiluminescent 1,2-dioxetanes have assumed increasing importance inrecent years, particularly with the advent of the enzymaticallycleavable chemiluminescent 1,2-dioxetanes disclosed in Bronstein U.S.patent application Ser. No. 889,823, filed Jul. 24, 1986 (the "'823application"); Bronstein, et al. U.S. patent application Ser. No.140,035, filed Dec. 31, 1987; Edwards U.S. patent application Ser. No.140,197, filed Dec. 31, 1987 (the "'197 application") and Edwards, etal. U.S. patent application Ser. No. 213,672 ("'672 application"), filedJun. 30, 1988.

Again in marked contrast to enzymatically cleavable 1,2-dioxetanes, thevarious chemically cleavable chemiluminescent 1,2-dioxetanes known up tonow have had little if any utility as reporter molecules in any type ofanalytical technique, and certainly not in bioassays. This is becausethe known chemically cleavable compounds are for the most part waterinsoluble--except for certain acetoxy-substituted 1,2-dioxetanes thatare somewhat water-soluble as well as organic solvent-soluble--and thusmay not be useful in biological assays unless they could somehow bemodified by adding to them groups or substituents that allow conjugationto a biological species, e.g., an antibody, thus permitting suchconjugated chemically cleavable 1,2-dioxetanes to be used as chemicallyactivated chemiluminogenic labels.

The water solubility of typical enzymatically cleavable chemiluminescent1,2-dioxetanes, on the other hand--e.g., adamantyl-appendedenzymatically cleavable 1,2-dioxetanes that decompose in the presence ofa suitable enzyme with light emission, such as 3-(4-methoxyspiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decan!-4-yl)phenyl phosphate andits salts, e.g., the disodium salt, identified hereinbelow in shorthandfashion as adamantylidenemethoxyphenoxyphosphorylated dioxetane("AMPPD"), and 3-(4-methoxyspiro 1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7!decan!-4-yl)phenyloxy-3"-β-D-galactopyranoside and its salts("AMPGD")--makes them eminently suitable for use as reporter moleculesin many types of analytical techniques carried out in aqueous media, andespecially in bioassays.

It has been observed that AMPPD in aqueous solution, and also in thepresence of chemiluminescence enhancers, e.g., a polymeric ammonium,phosphonium or sulfonium salt such as polyvinylbenzyl(benzyldimethylammonium chloride)! ("BDMQ") and otherheteropolar polymers (see Voyta, et al. U.S. patent application Ser. No.203,263, filed Jun. 1, 1988), exhibits longer than optimum periods oftime to reach constant light emission characteristics ("t1/2", definedas the time necessary to attain one half of the maximumchemiluminescence intensity at constant, steady-state light emissionlevels; this emission half-life varies as a function of the stability ofthe dioxetane oxyanion in various environments).

Statistically, approximately seven t1/2 periods are required to reachsteady-state light emission kinetics. The t1/2 for AMPPD atconcentrations above 2×10⁻⁵ M in aqueous solution at pH 9.5 in thepresence of BDMQ has been found to be 7.5 minutes. At 4×10⁻³ M in theabsence of BDMQ the t1/2 has been found to be approximately 30-60minutes, while at 2×10⁻⁵ M in aqueous solution the t1/2 for AMPPD hasbeen found to be 2.5 minutes.

In rapid bioassays that employ enzymatically cleavable chemiluminescent1,2-dioxetanes as reporter molecules it is desirable to reachsteady-state light emission kinetics as quickly as possible so as todetect an "endpoint" in the assay. And while chemiluminescence intensitycan be measured before achieving steady-state kinetics, sophisticated,thermally controlled luminometry instrumentation must be used if onewishes to acquire precise data prior to steady-state emission kinetics.

Furthermore, AMPPD, in aqueous buffered solution both in the presenceand the absence of chemiluminescence enhancers such as BDMQ, exhibitshigher than desirable thermal and nonenzymatically activated lightemission, or "noise". Such noise can be attributed to emissions from theexcited states of adamantanone and of the methyl m-oxybenzoate anionderived from the aromatic portion of the AMPPD molecule. This noise canlimit the levels of detection, and thus prevent the realization ofultimate sensitivity, as the measured noise level of AMPPD isapproximately two orders of magnitude above the dark current in astandard luminometer.

Enzymatic cleavage of AMPPD with alkaline phosphatase also generatesanionic, dephosphorylated AMPPD--adamantylidenemethoxymethylphenolatedioxetane, or "AMP⁻ D". This phenolate anion can also be formedhydrolytically in small amounts, giving rise to a backgroundchemiluminescence signal which, in an organized molecular assembly, suchas a micelle, liposome, lamellar phase, thin layer, lipid bilayer,liposome vesicle, reversed micelle, microemulsion, microgel, latex,membrane or polymer surface, and in a hydrophobic environment such asthat produced by a chemiluminescence enhancer, e.g., BDMQ, can generatestrong, enhanced levels of light emission, thereby creating highbackground signals and substantially lowering the dynamic range of thesignal resulting from enzymatic hydrolysis of AMPPD.

Consistent with the above-described observations, we have postulated thefollowing mechanisms.

In the presence of enhancing polymers such as BDMQ:

1. Enzymatic Pathway (AP present): ##STR2##

2. Thermal Pathway (no AP present): ##STR3##

Even in the absence of enhancing polymer, AMPPD can exist in aqueoussolution as an aggregate:

3. Enzymatic Pathway: ##STR4##

4. Thermal and Hydrolytic Pathway: ##STR5##

In the above mechanisms, n>>>m; n and m are a function of the presenceor absence of enhancing polymer, and AMPPD concentration.

The excited state of the adamantanone singlet in aggregate form (n orm>1) may exhibit higher yields of signal emission, here againparticularly if "stabilized" to emit more light, as by the presence of achemiluminescence enhancer such as BDMQ, than does the excited energystate of unaggregated adamantanone. This is perhaps due to the former'shaving lower singlet states, lower yields of intersystem crossing orslower intersystem crossing than the latter, or to other as yet unknownfactors. Since luminometers, generally, are designed to detect allphotons emitted regardless of their energy, or their wavelength, 415 nmand 477 nm chemiluminescence are both detected as background noiseemissions. Similarly, when photographic or X-ray film is used to recordchemiluminescence, no discrimination between the different wavelengthemissions can easily be made, thus, sensitivity of detection is limitedby background noise.

Finally, the observed aggregation of AMPPD under the conditionsdescribed above may result from the amphiphilic nature of AMPPD, or itsphenolate anion, and like molecules: ##STR6##

It is, therefore, an object of this invention to decrease the timenecessary to conduct assays, and particularly bioassays, in whichenzymatically cleavable chemiluminescent 1,2-dioxetanes are used asreporter molecules.

It is also an object of this invention to provide new and improvedenzymatically cleavable chemiluminescent 1,2-dioxetanes which, when usedas reporter molecules in assays, and particularly bioassays, reduce thetime required to complete the assay.

A further object of this invention is to provide new and improvedenzymatically cleavable chemiluminescent 1,2-dioxetanes for use assubstrates for enzyme-based assays, and particularly bioassays, whichprovide improved signal to background behavior and thus provide improveddetection levels.

A still further object of this invention is to provide novelintermediates useful in synthesizing these improved enzymaticallycleavable 1,2-dioxetanes.

Another object of this invention is to provide methods of preparingthese enzymatically cleavable chemiluminescent 1,2-dioxetanes andintermediates therefor.

These and other objects, as well as the nature, scope and utilization ofthis invention, will become readily apparent to those skilled in the artfrom the following description and the appended claims.

SUMMARY OF THE INVENTION

This invention provides a new class of stable, enzymatically cleavablechemiluminescent 3-(substituted adamant-2'-ylidene)-1,2-dioxetanecompounds capable of reacting in aqueous media, e.g., in a sample ofbiological fluid in solution or on a solid surface, e.g., a membranesurface such as a nylon membrane, with an enzyme or enzyme modifiedspecific binding pair to release optically detectable energy.

In aqueous media these modified admantylidene dioxetanes enable assaysin which they are used as reporter molecules to be conducted faster andwith greater sensitivity than hitherto possible using AMPPD.

While we do not wish to be bound by any mechanism or theory advanced toexplain this unexpectedly superior behavior, it may be that the presenceof substituents of the type disclosed herein on or in the adamantylidenemoiety prevents the dioxetane molecules from packing efficiently, andthus prevents them from forming "stabilized" organized assemblies,whether in the form of micelles or some other aggregated state. Certainof these substituents may also hydrogen bond to other substances intheir aqueous environment, including water itself, thereby furtherpreventing aggregate formation. And, it is also possible that electronicand dipole effects may contribute to this phenomenon, as evidenced byshorter t1/2's and lower background noise.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 show TSH, RLV v. TSH for each of AMPPD and its bromo-,B-hydroxy-,A-hydroxy- and chloroadamant-2'-ylidene analogs,respectively, obtained as described in Example XII below.

FIGS. 6-10 compare the total luminescence emissions obtained from AMPPDand its A-hydroxy-, B-hydroxy, chloro- and bromoadamant-2'-ylideneanalogs, respectively, obtained as described in Example XIII below.

FIG. 11 shows the improved chemiluminescence intensity obtained usingthe chloroadamant-2'-ylidene analog of AMPPD as compared to AMPPD itselfas the reporter molecule in a nucleic acid assay; see Example XIV below.

FIG. 12 shows the kinetics of the light emissions obtained using thechloroadamant-2'-ylidene analog of AMPPD and AMPPD itself as thereporter molecules in a nucleic acid assay; see Example XVI below.

FIG. 13 is the dose response curve at five minutes for alkalinephosphatase dilution with the chloroadamant-2'-ylidene analog of AMPPDplus poly vinyl(benzyldimethylammonium chloride)! ("BDMQ"), compared tothe dose response curve at five minutes for AMPPD itself plus BDMQ; seeExample XIX below.

FIG. 14 is the dose response curves at 20 minutes for the samesubstances whose dose response curves are shown in FIG. 13.

FIG. 15 is the dose response curve at five minutes for alkalinephosphatase dilution with the chloroadamant-2'-ylidene analog of AMPPDplus BDMQ-fluorescein ("emerald"), compared to the dose response curveat five minutes for AMPPD itself plus emerald; again see Example XIXbelow.

FIG. 16 is the dose response curves at 20 minutes for the samesubstances whose dose response curves are shown in FIG. 15.

FIG. 17 shows a TPA sequence's images, obtained as described in ExampleXX below using (1) AMPPD and (2) AMPPD's chloroadamant-2'-ylideneanalog.

DETAILED DESCRIPTION OF THE INVENTION

The novel chemiluminescent 3-(substituted adamant-2'-ylidene)1,2-dioxetanes of this invention can be represented by the generalformula: ##STR7##

In Formula I, X and X¹ each represent, individually, a substituent atthe 5' and 7' positions on the adamant-2'-ylidene substituent which canbe hydrogen, a hydroxyl group (a slightly electron withdrawing groupwhen hydrogen-bonded to water), a halo substituent, i.e., fluoro orchloro (electron withdrawing groups) or bromo or iodo (polarizable,mesomeric groups), an unsubstituted straight or branched chain loweralkyl group, preferably methyl; a substituted straight or branched chainlower alkyl group monosubstituted or having two or more substituentswhich can be the same or different, e.g., a hydroxyalkyl group such as ahydroxymethyl group, a haloalkyl group such as trifluoromethyl, and thelike; an unsubstituted aryl group, preferably a phenyl group; asubstituted aryl group, preferably one whose aryl ring contains sixcarbon atoms monosubstituted or having two or more substituents whichcan be the same or different, e.g., a halo substituent, as inp-bromophenyl or p-chlorophenyl, an alkoxy substituent, e.g.,p-methoxyphenyl (an electron donating group), a hydroxyalkoxysubstituent, e.g., hydroxyethoxy or hydroxypropoxy, a cyano group, or anamide group, e.g., a formamido or acetamido group, and the like, withthe proviso that at least one of X and X¹ is other than hydrogen.

Also included within the scope of the novel chemiluminescent3-(substituted adamant-2'-ylidene) 1,2-dioxetanes of this invention arecompounds otherwise similar to those of Formula I above which, insteadof bearing a substituent other than hydrogen at the 5' or 7', or 5' and7' positions, are instead substituted at the 4' position with amethylene group.

When the adamantylidene group is monosubstituted with one of theforegoing substituents other than hydrogen, a mixture of the syn- andanti-isomers will be obtained when such compounds are synthesized. Incertain cases, e.g., for monoiodo-substituted adamantylidene dioxetanes,one isomer will exhibit greater chemiluminescence intensity ondecomposition than the other. In other cases, e.g., formonohydroxy-substituted adamantylidene dioxetanes, the two isomers willbe equivalent or nearly so in chemiluminescence properties, such asintensity. In either case, the isomers may be separated before beingused, e.g., as reporter molecules in bioassays, by techniques such asthose disclosed in Edwards, et al. U.S. patent application Ser. No.244,006, filed Sep. 14, 1988, or they may be used as chromatographed,isomeric mixtures without separation.

Dioxetanes whose adamantylidene substituents are further substitutedwith two of the foregoing substituents other than hydrogen (X andX'≠hydrogen) do not exhibit syn/anti isomerism, although, of course,position isomers are possible when two different substituents arepresent, e.g., 5'-hydroxy-7'-chloro- and5'-chloro-7'-hydroxy-substituted adamantylidene dioxetanes.

The symbols R₁ and R₂ can represent any of the substituents on the4-carbon atom of the dioxetane ring disclosed in the aforementionedBronstein; Bronstein, et al.; Edwards; Edwards, et al. and Voyta, et al.applications, so long as when R₁ and R₂ represent individualsubstituents the R₂ substituent is aromatic, heteroaromatic, or anunsaturated substituent in conjugation with an aromatic ring, and atleast one of R₁ and R₂ is, or R₁ and R₂ taken together are, anenzymatically cleavable labile group-substituted fluorescent chromophoregroup that produces a luminescent substance when the enzymaticallyremovable labile substituent thereof is removed by an enzyme.

Thus, for example, the symbol R₁ can represent hydrogen, or a bond whenR₂ represents a substituent bound to the dioxetane ring through a spirolinkage, or an organic substituent that does not interfere with theproduction of light and that satisfies the valence of the dioxetane ringcarbon atom to which it is attached to result in a tetravalent dioxetanering carbon atom, such as an alkyl, aryl, aralkyl, alkaryl, heteroalkyl,heteroaryl, cycloalkyl or cycloheteroalkyl group, e.g., a straight orbranched chain alkyl group having from 1 to 7 carbon atoms, inclusive; astraight or branched chain hydroxyalkyl group having from 1 to 7 carbonatoms, inclusive, an --OR group in which R is a C₁ -C₂₀ unbranched orbranched, unsubstituted or substituted, saturated or unsaturated alkyl,cycloalkyl, cycloalkenyl, aryl, aralkyl or aralkenyl group, any of whichmay additionally be fused to R₂ such that the emitting fragment containsa lactone ring or an N, O or S heteroatom-containing group, or an enzymecleavable group bonded directly to the 4-carbon atom of the dioxetanering, or to one of the other aforementioned R₁ groups, that contains abond cleavable by an enzyme to yield either directly or by subsequentadjustment of pH an electron-rich moiety, e.g., an oxygen anion, asulfur anion or a nitrogen anion (the latter being, for example, anoxime or an amido anion such as a sulfonamido anion) bonded to thedioxetane ring. Preferably, R₁ is an alkoxy group, and especially amethoxy group, when R₂ is singly bonded to the dioxetane's 4-carbonatom.

The symbol R₂ can also represent any organic substituent that does notinterfere with the production of light and that satisfies the valence ofthe 4-carbon atom of the dioxetane ring to which it is attached.Preferably, R₂ will represent any of a number of light-emittingfluorophore-forming fluorescent chromophore groups that permit thecorresponding dioxetane decomposition fragments to absorb energy andform an excited state from which they emit optically detectable energyto return to their ground state, substituted with an enzyme cleavablegroup that contains a bond cleavable by an enzyme to yield eitherdirectly or by subsequent adjustment of pH an electron-rich moiety,again, for example, an oxygen anion, a sulfur anion or a nitrogen anion,bonded to the dioxetane ring.

Thus, for example, the symbol R₂ can represent, alone (or together withthe symbol R₁ to give a substituent spiro bonded to the 4-carbon atom ofthe dioxetane ring) fluorescent chromophore groups such as:

phenyl and phenyl derivatives;

naphthalene and naphthalene derivatives e.g.,5-dimethylaminonaphthalene-1-sulfonic acid and hydroxy naphthalenes;

anthracene and anthracene derivatives, e.g., 9,10-diphenylanthracene,9-methylanthracene, 9-anthracene carboxaldehyde, anthryl alcholols and9-phenylanthracene;

rhodamine and rhodamine derivatives, e.g., rhodols, tetramethylrhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine,diphenyldiethyl rhodamine and dinaphthyl rhodamine;

fluorescein and fluorescein derivatives, e.g., 5-iodoacetamidofluorescein, 6-iodoacetamido fluorescein and fluorescein-5-maleimide;

coumarin and coumarin derivatives, e.g.,7-dialkylamino-4-methylcoumarin, 4-bromomethyl-7-methoxycoumarin and4-bromomethyl-7-hydroxy coumarin;

erythrosin and erythrosin derivatives, e.g., hydroxy eyrthrosins,erythrosin-5-iodoacetamide and erythrosin-5-maleimide;

acridine and acridine derivatives, e.g., hydroxy acridines and 9-methylacridine;

pyrene and pyrene derivatives, e.g., N-(1-pyrene) iodoacetamide, hydroxypryrenes and 1-pyrenemethyl iodoacetate;

stilbene and stilbene derivatives, e.g., 6,6'-dibromostilbene andhydroxy stilbenes;

nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g., hydroxynitrobenzoxadiazoles, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole,2-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)methylaminoacetaldehyde and6-(7'-nitrobenz-2-oxa-1,3-diazole-4-yl)aminohexanoic acid;

quinoline and quinoline derivatives, e.g., 6-hydroxyquinoline and6-aminoguinoline;

acridine and acridine derivatives, e.g., N-methylacridine andN-phenylacridine;

acidoacridine and acidoacridine derivatives, e.g., 9-methylacidoacridineand hydroxy-9-methylacidoacridine;

carbazole and carbazole derivatives, e.g., N-methylcarbazole andhydroxy-N-methylcarbazole;

fluorescent cyanines, e.g., DCM (a laser dye), hydroxy cyanines,1,6-diphenyl-1,3,5-hexatriene, 1-(4-dimethylaminophenyl)-6-phenylhexatriene and the corresponding 1,3-butadienes;

carbocyanines and carbocyanine derivatives, e.g., phenylcarbocyanine andhydroxy carbocyanines;

pyridinium salts, e.g., 4(4-dialkyldiaminostyryl)-N-methyl pyridiniumiodate and hydroxy-substituted pyridinium salts;

oxonols; and

resorofins and hydroxy resorofins.

The symbol R₂, together with the symbol R₁, can also represent a fusedfluorescent chromophore group, bonded to the 4-carbon atom of thedioxetane ring through a spiro linkage, having the general formula:##STR8##

In this formula Y is ##STR9## --O--, --S--, or --NR₆, where each of R₄,R₅ and R₆, independently, is hydrogen, a branched or straight chainalkyl group having 1 to 20 carbon atoms, inclusive, e.g., methyl,n-butyl or decyl, a branched or straight chain heteroalkyl group having1 to 7 carbon atoms, inclusive, e.g., methoxy, hydroxyethyl orhydroxypropyl; an aryl group having 1 or 2 rings, e.g, phenyl, naphthylor anthryl; a heteroaryl group having 1 or 2 rings, e.g., pyrrolyl orpyrazolyl; a cycloalkyl group having 3 to 7 carbon atoms, inclusive, inthe ring, e.g., cyclohexyl; a heterocycloalkyl group having 3 to 6carbon atoms, inclusive, in the ring, e.g., dioxane; an aralkyl grouphaving 1 or 2 rings, e.g., benzyl; or an alkaryl group having 1 or 2rings, e.g., tolyl; and each R₃, independently, can be hydrogen; anelectron-withdrawing group, such as a perfluoroalkyl group havingbetween 1 and 7 carbon atoms, inclusive, e.g., trifluoromethyl; ahalogen; CO₂ H, --ZCO₂ H, --SO₃ H, ZSO₃ H, --NO₂, --ZNO₂, --C.tbd.N, or--Z₁ C.tbd.N, where Z is a branched or straight chain alkyl group having1 to 7 carbon atoms, inclusive, e.g., methyl; an aryl group having 1 or2 rings, e.g., phenyl; an electron-donating group, e.g., a branched orstraight chain C₁ -C₇ alkoxy group, e.g., methoxy or ethoxy; an aralkoxygroup having 1 or 2 rings, e.g., phenoxy; a branched or straight chainC₁ -C₇ hydroxyalkyl group, e.g., hydroxymethyl or hydroxyethyl; ahydroxyaryl group having 1 or 2 rings, e.g., hydroxyphenyl; a branchedor straight chain C₁ -C₇ alkyl ester group, e.g., acetate; an aryl estergroup having 1 or 2 rings, e.g., benzoate; or a heteroaryl group having1 or 2 rings, e.g., benzoxazole, benzthiazole, benzimidazole orbenztriazole. Furthermore, two or more of the R₃ groups can form a fusedring or rings which can themselves be unsubstituted or substituted.

The symbol R₂, alone or together with the symbol R₁, can likewiserepresent a particular class of fused polycyclic ring-containingfluorophore moieties having a labile ring substituent containing a bondwhich, when cleaved, renders the fused polycyclic moiety electron-richto in turn render the dioxetane compound decomposable to emit light. Themembers of this class are those in which the labile ring substituent'spoint of attachment to the fused polycyclic ring, in relation to thisring's point(s) of attachment to the dioxetane ring (single bondattachment or, when R₁ represents a bond, a spiro linkage), is such thatthe total number of ring sp² atoms separating these points ofattachment, including the sp² ring carbon atoms at the points ofattachment, is an odd whole number; see the Edwards, et al. '672application.

Included among the fused polycyclic ring compounds whose residues can beused to form this fluorophore moiety are the fused polycyclic aromatichydrocarbon ring fluorophoric compounds mentioned above, andparticularly ones containing from 9 to about 30 ring carbon atoms,inclusive, such as naphthalene: ##STR10## the substituent bondsindicating a 1,6-substitution pattern as in disodium 6-(4-methoxyspiro1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo 3.3.1.1³,7!decan!-4-yl)-1-naphthalenyl phosphate, pentalene, azulene, heptalene,as-indacene, s-indacene, biphenylene, perylene, acenaphthylene,phenanthrene, anthracene, acephenanthrylene, aceanthrylene,triphenylene, pyrene, chrysene, naphthacene, and the like, as well asderivatives thereof substituted with one or more non-labile substituentssuch as those mentioned above as being represented by the symbols R₃, R₄and R₅.

The fused polycyclic ring portion of such "odd pattern substituted"fluorophore moieties represented by R₂ alone or together with R₁ canalso be the residues of nonaromatic, i.e., less than fully aromatic,fused polycyclic hydrocarbon ring fluorophoric compounds, having alabile ring substituent containing a bond which, when cleaved, rendersthe fused, less than fully aromatic polycyclic moiety electron-rich toin turn render the dioxetane compound decomposable to emit light,unsubstituted or substituted with one or more of the aforementionednon-labile substituents, and containing from 10 to about 30 ring carbonatoms, inclusive, such as fluorene, 3,4-dihydro-3,3-dimethylnaphthalene,dibenzosuberene, 9,10-dihydrophenanthrene, indene, indeno 1,2-a! indene,phenalene, fluoroanthrene, and the like.

Further, the fused polycyclic ring portion of fluorophore moietiesrepresented by R₂ alone or together with R₁ can also be the residue of afused polycyclic heteroaromatic or less than fully aromatic fused ringheterocyclic fluorophore-forming group, e.g., dibenzothiophene,dibenzofuran, 2,2-dimethyl-2H-chromene, xanthene, piperidine, quinoline,isoquinoline, phenanthridine, carbostyryl, phenoxazine, phenothiazine,phenanthroline, purine, phthalazine, naphthyridine, N-acylindole,chroman, isochroman, N-acylindoline, isoindoline, and the like,unsubstituted or substituted with one or more of the aforementionednon-labile substituents, and containing from 9 to about 30 ring atoms,inclusive, the bulk of which are carbon atoms.

A preferred enzymatically removable group with which at least one of R₁and R₂ is substituted is a phosphate group, particularly a phosphateester group represented by the general formula: ##STR11## wherein M+represents a cation such as alkali metal, e.g., sodium or potassium,ammonium, or a C₁ -C₇ alkyl, aralkyl or aromatic quaternary ammoniumcation, N(R₇)+₄, in which each R₇ can be alkyl, e.g., methyl or ethyl,aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g.,pyridinium. The disodium salt is particularly preferred. Such phosphateester groups can be cleaved using an enzyme such as alkaline phosphataseto produce oxygen anion-substituted groups that will, in turn,destabilize the dioxetane with rupture of its oxygen-oxygen bond toproduce light. The quaternary ammonium cations in such phosphate estergroups can also be connected through one of their guaternizing groups toa polymeric backbone, viz. ##STR12## where n is greater than 1, or canbe part of a polyquaternary ammonium salt, i.e., an ionene polymer.

Another preferred enzymatically removable group is the β-D-galactosidegroup, which can be cleaved with the enzyme β-D-galactosidase to yieldthe conjugate acid of the dioxetane phenolate, which upon pH adjustmentchemiluminesces.

Enzymatically cleavable substituents that can be used also includeenzyme-cleavable alkanoyloxy groups, e.g., an acetate ester group, or anenzyme-cleavable oxacarboxylate group, 1-phospho-2,3-diacylglyceridegroup, 1-thio-D-glucoside group, adenosine triphosphate analog group,adenosine diphosphate analog group, adenosine monophosphate analoggroup, adenosine analog group, α-D-galactoside group, β-D-galactosidegroup, α-D-glucoside group, β-D-glucoside group, α-D-mannoside group,β-D-mannoside group, β-D-fructofuranoside group, β-D-glucosiduronategroup, p-toluenesulfonyl-L-arginine ester group orp-toluenesulfonyl-L-arginine amide group.

The substituted adamant-2-ylidene moiety spiro bonded to the 3-carbonatom of the dioxetane ring, illustrated in formula I above, can bereplaced by other similarly substituted fused polycycloalkylidene groupshaving two or more fused rings, each ring having from 3 to 12 carbonatoms, inclusive, such as bicyclo 3.3.1.!nonan-9-ylidene, hexacyclo-5.5.1.0.²,6 0.³,10 0.⁴,8 0⁹,13 !tridecan-5-ylidene, pentacyclo5.4.0.0.²,6 0.³,10 0⁵,9 !undecan-4-ylidene, and the like.

PCT Application No. WO88/00695, published Jan. 28, 1988 based on theBronstein '823 application, discloses enzymatically cleavable1,2-dioxetanes which are substituted at the 3-carbon atom with a definedsubstituent "T-V" and at the 4-carbon atom with defined substituents "X"and "Y-Z". This published application also discloses, at p.3,1s.6-12,that:

In preferred embodiments, one or more of groups T, X, or Y furtherinclude a solubilizing substituent, e.g., carboxylic acid, sulfonicacid, or quaternary amino salt; group T of the dioxetane is apolycycloalkyl group, preferably adamantyl; the enzyme-cleavable groupincludes phosphate; and the enzyme possesses phosphatase activity,

at p.22,1.33-p.23,1.6 that:

For example, the enzyme-cleavable group Z can be bonded to group X ofthe dioxetane, instead of group Y. The specific affinity substance canbe bonded to the dioxetane through groups X, Y, or T (preferably groupX), instead of the enzyme. In this case, the group to which the specificaffinity substance is bonded is provided with, e.g., a carboxylic acid,amino or maleimide substituent to facilitate bonding,

and at p.23,1s.11-21 that:

Groups X, Y, or T of the dioxetane can be bonded to a polymerizablegroup, e.g., a vinyl group, which can be polymerized to form ahomopolymer or copolymer.

Groups X, Y, or T of the dioxetane can be bonded to, e.g., membranes,films, beads, or polymers for use in immuno- or nucleic acid assays. Thegroups are provided with, e.g., carboxylic acid, amino, or maleimidesubstituents to facilitate bonding.

Groups X, Y, or T of the dioxetane can contain substituents whichenhance the kinetics of the dioxetane enzymatic degradation, e.g.,electron-rich moieties (e.g., methoxy).

Groups Y and T of the dioxetane, as well as group X, can containsolubilizing substituents.

The problem solved by the 3-(substituted adamant-2'-ylidene)1,2-dioxetane compounds disclosed and claimed herein is not addressed inthis published PCT application, nor are these compounds themselvesdisclosed in this or any other reference of which the inventors areaware.

The overall synthesis of these 3-(substitutedadamant-2'-ylidene)-1,2-dioxetanes can be accomplished using methodssuch as those disclosed in the aforementioned Bronstein and Edwardsapplications and in Edwards, et al. U.S. patent application Ser. No.279,176 ("'176 application"), filed Sep. 6, 1989. Thus, for example,1,2-dioxetanes coming within the scope of formula I above in which Xrepresents a hydroxyl group, X¹ represents hydrogen, R₁ represents amethoxy group and R₂ represents a phosphoryloxy salt-substituted phenylgroup, preferably a meta-phosphoryloxy salt-substituted phenyl group,can be synthesized in accordance with the methods disclosed in the '176application by a reaction sequence that can be illustrated schematicallyas follows: ##STR13##

As specifically exemplified below, the ##STR14## starting material can,if desired, be reacted with an orthoformate such astrimethylorthoformate, methanol, and p-toluenesulfonic acid, to give theintermediate: ##STR15## which, when then reacted with (R₈ O)₃ P andLewis acid, gives the phosphonate ester intermediate: ##STR16##

In the foregoing reaction sequence R₈ represents a lower alkyl group,e.g., methyl, ethyl or butyl. R₉ represents an acyl group containingfrom 2 to about 14 carbon atoms, inclusive, such as acetyl, propionyl,mesitoyl or pivaloyl, Q represents a halogen, e.g., chloro or bromo, orOR₈, and M represents, independently, a proton, a metal cation, e.g.,Na⁺, or K⁺, or an ammonium, substituted ammonium, quaternary ammonium or(H⁺) pyridinium cation. Thiolate cleavage as described in the Edwards'197 application can be used in place of base cleavage of the OR₉ groupin step 5 of the reaction sequence illustrated above, in which case R₉can be a lower alkyl, lower alkenyl or aralkyl group, e.g., methyl,allyl or benzyl. The product of base or thiolate cleavage can have, inplace of R₉, hydrogen or an alkali metal cation, e.g., lithium, sodiumor potassium; see copending Edwards, et al. U.S. patent application Ser.No. 07/574,789, filed of even date herewith.

The intermediates represented above by the formula: ##STR17## where onlyone of X and X¹ is other than hydrogen are known compounds, or arereadily synthesizable from known starting materials using art-recognizedmethods. For example, in the case of the monosubstitutedadamantan-2-ones (one of X and X¹ is hydrogen):

-5-hydroxyadamantan-2-one is prepared as described in Geluk, Synthesis,374 (1972);

-5-bromoadamantan-2-one and 5-chloroadamantan-2-one are prepared asdescribed in Geluk, et al., Tetrahedron, 24, 5369 (1968).

Where X or X¹ is fluoro, unsubstituted (lower) alkyl, e.g., t-butyl,substituted (lower) alkyl, e.g., trifluoromethyl, unsubstituted aryl,e.g., phenyl, or substituted aryl, e.g., p-chlorophenyl, p-methoxyphenylor p-nitrophenyl, see 1e Noble, et al., J. Am. Chem. Soc., 108 1598(1986) and Walborsky, et al., J. Am. Chem. Soc., 109, 6719 (1987) (X orX¹ =hydroxymethyl).

Simple unit processes allow the conversion of several of theabovementioned X or X¹ substituents, or others known in the art, to 5-X-or 5-X¹ -adamantane-2-ones where X or X¹ may be trialkylsilyloxy, iodoor cyano groups. These moieties are stable under the mild conditionsused in step 4 of the foregoing reaction sequence. For example, when5-hydroxyadamantan-2-one is refluxed for 7 hours with 57% hydriodicacid, 5-iodoadamantan-2-one (m.p. 73°-76° C.) is obtained.5-Carboxyadamantan-2-one, prepared as described in Lantvoev,J.Obshch.Khim., 12, 2361 (1976) or Le Noble, et al., J.Org.Chem., 48,1101 (1983), after saponification of the methyl ester, can be convertedto 5-cyanoadamantan-2-one by the three step procedure of Tabushi, etal., J.Org.Chem., 38, 3447 (1973), used for access to the isomeric1-cyanoadamantan-2-one through the intermediacy of a keto amide.5-Trimethylsilyloxyadamantan-2-one (m.p. 34°-38° C.), useful as aprotected version of 5-hydroxyadamantan-2-one, allows the use of onlyone equivalent of base in step 4 of the foregoing reaction sequence toprepare the corresponding enol ether, which can then be desilylatedusing standard techniques.

As will be appreciated by one skilled in the art, other X and X¹ groupsneed not be static during the entire reaction sequence, but may betransformed by reactions which are compatible with other structuralconsiderations at any stage. For example, it has been discovered thatwhen X or X¹ is a chlorine or a bromine atom, enol ether intermediatesproduced in steps 4 and 5 of the foregoing reaction sequence are subjectto facile solvolysis in the presence of molar excesses of diols orliquid ammonia in a bomb at high temperature. The reaction rate withdiols such as ethylene glycol or propylene glycol becomes appreciableonly at elevated temperatures (105°-120° C.) in the presence of a protonacceptor such as potassium carbonate. In general, this reaction is slow,but clean, and avoids the use of silver or heavy metal salts often usedto stimulate hydroxyalkyl ether formation.3-(Methoxy-5-(2-hydroxyethoxy)tricyclo- 3.3.1.1³,7!dec-2-ylidenemethyl)phenol can be esterified with trimethylacetylchloride and triethylamine to give the corresponding diester, which canthen be selectively cleaved, using potassium carbonate in methanol, togive the phenolic monoester. The ensuing phosphorylation step,incorporating simultaneous β-elimination and saponification of thehindered ester with sodium methoxide in methanol, will furnish thehydroxyethoxy enol ether phosphate.

Reaction with liquid ammonia in dioxane, under pressure, to give3-(methoxy-5-aminotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenol fromthe corresponding 5-bromo compound is carried out using the proceduredescribed by Hummelen, Dissertation, University of Groningen, TheNetherlands, p. 60 (1985). Immediate acylation of the thus-obtainedamino enol ether phenol, using two equivalents of acetyl chloride oracetic formic anhydride and 4-dimethylaminopyridine as the base,following the procedure of Gawronski, et al., J. Am. Chem. Soc., 109,6726 (1987) used for the esterification of(5-hydroxyadamantylidene)ethanol, will give the formamido or acetamidophenolic esters, which can be selectively saponified as described supraand then phosphorylated and photooxygenated as described infra.

Meijer, Dissertation, University of Groningen, The Netherlands (1982);Numan, et al., J.Org.Chem., 43, 2232 (1978); and Faulkner, et al.,J.Chem.Soc., Chem.Comm., 3906 (1971), provide access to4-methyleneadamantan-2-one (X and X¹ =hydrogen, methylene at the 4'position in Formula I above) as the starting material for step 4 of theforegoing reaction sequence and subsequent reaction with a suitablephosphonate-stabilized carbanion. The difference in the reactivity ofsinglet oxygen toward the enol ether instead of the exomethylenefunction ensures that disodium 3-(4-methoxyspiro-1,2-dioxetane-3,2'-(4'-methylene)tricyclo 3.3.1.1³,7!decan!-4-yl-)phenyl phosphate will be obtained as the photooxygenationproduct.

If a selectively cleavable pivaloyloxyaryl enol ether will be obtainedin any of the reactions immediately preceding the addition of anenzyme-removable group such as a phosphate ester group, it will be moreconvenient to avoid isolation of the hydroxyaryl enol ether. This can beaccomplished by directly splitting the pivaloyl ester with oneequivalent of sodium methoxide in methanol and isolating the sodiumaryloxide species as a dry solid by removing all volatiles at theconclusion of the reaction. In such a case, step 6 of the foregoingreaction sequence will be run using this preformed salt in a dry, polaraprotic solvent such as dimethylformamide without using a Lewis base,and the inorganic salt by-products will be removed in the work-up phaseof step 7 or step 8.

The 2-cyanoethylphosphate diester product of step 7 undergoesbeta-elimination to the phosphate monoester in step 8. In step 8, thederivatives where X or X¹ =chlorine or bromine are preferably reactedwith a volatile amine such as ammonia, or with a solvent-soluble organicamine such as "DBU" (1,8-diazabicyclo 5.4.0!undec-7-ene) in an alcoholsolvent, e.g., methanol. The use of ammonia at atmospheric pressure orabove and at ambient temperatures is particularly advantageous, asexcess base is simply volatized in vacuo at the end of the reaction.

Oxidation of the enol ether phosphate in step 9 of the foregoingreaction sequence can be carried out photochemically, as indicated, byreaction with singlet oxygen (¹ O₂) in a halogenated solvent, e.g., ahalogenated hydrocarbon such as chloroform, which may also contain acosolvent, e.g., a lower alkanol such as methanol. Singlet oxygen can begenerated using a photosensitizer such as polymer-bound Rose Bengal(Polysciences, Inc.), methylene blue, or 5, 10, 15, 20-tetraphenyl-21H,23H-porphine ("TPP").

Alternatively, the crude 2-cyanoethyl phosphate diesters obtained instep 7 of the foregoing reaction sequence can be oxidized with singletoxygen to their 1,2-dioxetane counterparts. Subsequent reaction withsodium methoxide in methanol at room temperature, followed by aqueouswork-up and preparative reverse phase high pressure liquidchromatography, gives the pure 1,2-dioxetane phosphate monoester saltsas mixtures of their syn- and anti-isomers. Chemical methods of1,2-dioxetane formation, including ones usingtriethylsilylhydrotrioxide, phosphate ozonides or triarylamine radicalcation-mediated one electron oxidation in the presence of tripletoxygen, can also be used.

Starting materials of the formula: ##STR18## where both X and X¹ are thesame and are other than hydrogen, or intermediates from which suchstarting materials can be synthesized using art-recognized methods, arealso known. For example, the use of symmetrically substituted5,7-bis-X,X¹ -adamantan-2-ones in step 4 of the foregoing reactionsequence will result in symmetrical 1,2-dioxetanes which contain X andX¹ substituents in both a syn and anti relationship to the four-membereddioxetane ring. The quinone monoacetal-based synthesis strategy ofStetter, et al. provides access to 5,7-dihydroxyadamantan-2-one by wayof bicyclo 3.3.1!nonan-3,7-dione-9-ethyleneacetal; Stetter, et al.,Liebigs Ann. Chem., 1807 (1977); see also Hamill, et al., Tetrahedron,27, 4317 (1971). 5,7-Dihydroxyadamantan-2-one can be converted to5,7-dibromoadamantan-2-one or its 5,7-dichloro and 5,7-diiodo analogsusing 47% aqueous hydrobromic acid, thionyl chloride or 57% aqueoushydriodic acid under the conditions described in Geluk, et al., loc.cit. 5,7-Dialkyladamantan-2-ones, e.g., 5,7-dimethyl-adamantan-2-one,can be synthesized by the method of Kira, et al., J. Am. Chem. Soc.,111, 8256 (1989). Solvolysis of 3-(methoxy-5,7-dibromotricyclo3.3.3.1³,7 !dec-2-ylidenedimethyl)phenol with diols such as ethyleneglycol or 1,4-butanediol in the presence of potassium carbonate willalso furnish the corresponding symmetrical bis-hydroxyalkoxy-substituted1,2-dioxetanes upon following the modified route to the mono-substitutedderivatives described above.

When one wishes to take advantage of cooperative effects produced by anX group other than hydrogen that is different from an X¹ group alsopresent that is also other than hydrogen, particularly where advantagescan be obtained using such enzymatically cleavable 1,2-dioxetanes asisomeric mixtures, unsymmetrical 5,7(X,X¹)adamantan-2-ones will be usedin step 4 of the foregoing reaction sequence.5-Bromo-7-trifluoroadamantan-2-one can be prepared according to themethod disclosed in Sorochinskii, et al., Zh. Obshch. Khim., 17, 2339(1981). 5-Chloro-7-hydroxyadamantan-2-one and5-methyl-7-hydroxyadamantan-2-one have been described by Stetter, etal., loc. cit., and 5-bromo-7-hydroxyadamantan-2-one can be synthesizedfrom 7-methylenebicyclo 3.3.1!nonane-3,9-dione-9-ethylene acetalfollowing the procedure of Stetter, et al., by dissolving this compoundin anhydrous ethanol and saturating the solution at 0° C. with gaseoushydrogen bromide instead of the gaseous hydrogen chloride used to obtainthe corresponding chloro derivative.

Intermediates and methods for the synthesis of compounds of formula Iabove in which R₁ is other than lower alkoxy and R₂ is other thanphosphoryloxy salt-substituted phenyl are found in the abovementionedBronstein, Bronstein, et al., Edwards and Edwards, et al., ('672)applications.

This invention, as indicated above, is also directed to the use of itschemiluminescent, enzymatically cleavable substituted 1,2-dioxetanes inart-recognized assays, including assays for detecting enzymes insamples, to kits for use in such assays, and to like uses and means foraccomplishing such uses.

For example, when using this invention to detect an enzyme in a sample,the sample is contacted with a dioxetane bearing a group capable ofbeing cleaved by the enzyme being detected. The enzyme cleaves thedioxetane's enzyme cleavable group to form a negatively chargedsubstituent (e.g., an oxygen anion) bonded to the dioxetane. Thisnegatively charged substituent in turn destabilizes the dioxetane,causing the dioxetane to decompose to form a fluorescent chromophoregroup that emits light energy. It is this chromophore group that isdetected as an indication of the presence of the enzyme. By measuringthe intensity of luminescence, the concentration of the enzyme in thesample can also be determined.

A wide variety of other assays exist which use visually detectable meansto determine the presence or concentration of a particular substance ina sample. The above-described dioxetanes can be used in any of theseassays. Examples of such assays include immunoassays to detectantibodies or antigens, e.g., δ- or β-hCG; enzyme assays; chemicalassays to detect, e.g., potassium or sodium ions; and nucleic acidassays to detect, e.g., viruses (e.g., HTLV III or cytomegalovirus, orbacteria (e.g., E. coli), and certain cell functions (e.g., receptorbinding sites).

When the detectable substance is an antibody, antigen, or nucleic acid,the enzyme capable of cleaving the enzyme cleavable group of thedioxetane is preferably bonded to a substance having a specific affinityfor the detectable substance (i.e., a substance that binds specificallyto the detectable substance), e.g., an antigen, an antibody, or anucleic acid probe. Conventional methods, e.g., carbodiimide coupling,are used to bond the enzyme to the specific affinity substance; bondingis preferably through an amide linkage.

In general, assays are performed as follows. A sample suspected ofcontaining a detectable substance is contacted with a buffered solutioncontaining an enzyme bonded to a substance having a specific affinityfor the detectable substance. The resulting solution is incubated toallow the detectable substance to bind to the specific affinity portionof the specific affinity-enzyme compound. Excess specificaffinity-enzyme compound is then washed away, and a dioxetane having agroup cleavable by the enzyme portion of the specific affinity-enzymecompound is added. The enzyme cleaves the enzyme cleavable group,causing the dioxetane to decompose into two carbonyl compounds (e.g., anester, a ketone or an aldehyde). The chromophore to which the enzymecleavable group had been bonded is thus excited and luminesces.Luminescence is detected (using, e.g., a cuvette, or light-sensitivefilm in a camera luminometer, or a photoelectric cell or photomultipliertube), as an indication of the presence of the detectable substance inthe sample. Luminescence intensity is measured to determine theconcentration of the substance.

Examples of specific assays follow.

A. Assay for Human IgG

A 96-well microtiter plate is coated with sheep anti-human IgG (F(ab)₂fragment specific). A serum sample containing human IgG is then added tothe wells, and the wells are incubated for 1 hour at room temperature.

Following the incubation period, the serum sample is removed from thewells, and the wells are washed four times with an aqueous buffersolution containing 0.15M NaCl, 0.01M phosphate, and 0.1% bovine serumalbumin (pH 7.4).

Alkaline phosphatase bonded to anti-human IgG is added to each well, andthe wells are incubated for 1 hr. The wells are then washed four timeswith the above buffer solution, and a buffer solution of aphosphate-containing dioxetane of this invention is added. The resultingluminescence caused by enzymatic degradation of the dioxetane isdetected in a luminometer, or with photographic film in a cameraluminometer.

B. Assay for hCG

Rabbit anti-α-hCG is adsorbed onto a nylon-mesh membrane. A samplesolution containing hCG, e.g., urine from a pregnant woman, is blottedthrough the membrane, after which the membrane is washed with 1 ml of abuffer solution containing 0.15M NaCl, 0.01M phosphate, and 0.1% bovineserum albumin (pH 7.4).

Alkaline phosphatase-labeled anti-β-hCG is added to the membrane, andthe membrane is washed again with 2 ml of the above buffer solution. Themembrane is then placed in the cuvette of a luminometer or into a cameraluminometer, and contacted with a phosphate-containing dioxetane of thisinvention. The luminescence resulting from enzymatic degradation of thedioxetane is then detected.

C. Assay for Serum Alkaline Phosphatase

2.7 ml of an aqueous buffer solution containing 0.8M2-methyl-2-aminopropanol is placed in a 12×75 mm pyrex test tube, and0.1 ml of a serum sample containing alkaline phosphatase added. Thesolution is then equilibrated to 30° C. 0.2 ml of a phosphate-containingdioxetane of this invention is added, and the test tube immediatelyplaced in a luminometer to record the resulting luminescence. The levelof light emission will be proportional to the rate of alkalinephosphatase activity.

D. Nucleic Acid Hybridization Assay

A sample of cerebrospinal fluid (CSF) suspected of containingcytomegalovirus is collected and placed on a membrane, e.g., a nylon ornitrocellulose membrane. The sample is then chemically treated with ureaor guanidinium isothiocyanate to break the cell walls and the degradeall cellular components except the viral DNA. The strands of the viralDNA thus produced are separated and attached to the nitrocellulosefilter. A DNA probe specific to the viral DNA and labeled with alkalinephosphatase is then applied to the filter; the probe hybridizes with thecomplementary viral DNA strands. After hybridization, the filter iswashed with an aqueous buffer solution containing 0.2M NaCl and 0.1 mMTris-HCl (pH=8.10) to remove excess probe molecules. Aphosphate-containing dioxetane of this invention is added and theresulting luminescence from the enzymatic degradation of the dioxetaneis measured in a luminometer or detected with photographic film.

E. Assay for Galactosidase

In the assays described above and in the working examples to followdioxetanes containing α- or β-galactosidase-cleavable α-D- orβ-D-galactoside (galactopyranoside) groups, respectively, can be added,and the luminescence resulting from the enzymatic cleavage of the sugarmoiety from the chromophore measured in a luminometer or detected withphotographic film.

F. Electrophoresis

Electrophoresis allows one to separate complex mixtures of proteins andnucleic acids according to their molecular size and structure on gelsupports in an electrical field. This technique is also applicable toseparate fragments of protein after proteolysis, or fragments of nucleicacids after scission by restriction endonucleases (as in DNAsequencing). After electrophoretic resolution of species in the gel, orafter transfer of the separated species from a gel to a membrane, thebonds are probed with an enzyme bound to a ligand. For example, peptidefragments are probed with an antibody covalently linked to alkalinephosphatase. For another example, in DNA sequencing alkalinephosphatase--avidin binds to a biotinylated nucleotide base. Thereafter,an AMPPD analog of this invention is added to the gel or membranefilter. After short incubation, light is emitted as the result ofenzymatic activation of the dioxetane to form the emitting species. Theluminescence is detected by either X-ray or instant photographic film,or scanned by a luminometer. Multichannel analysis further improves theprocess by allowing one to probe for more than one fragmentsimultaneously.

G. Solid State Assays

In solid state assays, it is desirable to block nonspecific binding tothe matrix by pretreatment of nonspecific binding sites with nonspecificproteins such as bovine serum albumin (BSA) or gelatin. It has beenfound that some commercial preparations of BSA contain small amounts ofsubstances that exhibit phosphatase activity that will produceundesirable background chemiluminescence from AMPPD. It has also beendiscovered, however, that certain water-soluble synthetic macromolecularsubstances are efficient blockers of nonspecific binding in solid stateassays using dioxetanes. Preferred among such substances arewater-soluble polymeric quaternary ammonium salts such as BDMQ,poly(vinylbenzyltrimethylammonium chloride) (TMQ), andpoly(vinylbenzyltributylammonium chloride) (TBQ). Other such substancesare disclosed in the aforementioned Voyta, et al. '263 application andlisted in Table III below.

H. Assay for Nucleotidase

An assay for the enzyme ATPase is performed in two steps. In the firststep, the enzyme is reacted at its optimal pH (typically pH 7.4) with asubstrate comprising ATP covalently linked via a terminal phosphoesterbond to a chromophore-substituted 1,2-dioxetane to produce aphosphoryl-chromophore-substituted 1,2-dioxetane. In the second step,the product of the first step is decomposed by the addition of acid tobring the pH to below 6, preferably to pH 2-4, and the resulting lightmeasured in a luminometer or detected with chromatographic film. In asimilar two-step procedure, ADPase is assayed using as the substrate anADP derivative of a chromophore-substituted 1,2-dioxetane of thisinvention, and 5'-nucleotidase assayed using as the substrate anadenylic acid derivative of a chromophore-substituted 1,2-dioxetane ofthis invention. The second step can also be carried out by adding theenzyme alkaline phosphatase to decompose thephosphoryl-chromophore-substituted 1,2-dioxetane.

I. Nucleic Acid Sequencing

DNA or RNA fragments, produced in sequencing protocols, can be detectedafter electrophoretic separation using the chemiluminescent1,2-dioxetanes of this invention.

DNA sequencing can be performed by a dideoxy chain termination methodSanger, F., et al., Proc. Nat. Acad. Sci. (USA), 74:5463 (1977)!.Briefly, for each of the four sequencing reactions, single-strandedtemplate DNA is mixed with dideoxynucleotides and biotinylated primerstrand DNA. After annealing, Klenow enzyme and deoxyadenosinetriphosphate are incubated with each of the four sequencing reactionmixtures, then chase deoxynucleotide triphosphate is added and theincubation continued.

Subsequently, DNA fragments in reaction mixtures are separated bypolyacrylamide gel electrophoresis (PAGE). The fragments are transferredto a membrane, preferably a nylon membrane, and the fragmentscross-linked to the membrane by exposure to UV light, preferably ofshort wave length.

After blocking non-specific binding sites with a polymer, e.g., heparin,casein or serum albumin, the DNA fragments on the membrane are contactedwith avidin or streptavidin covalently linked to an enzyme specific forthe enzyme-cleavable group of the particular 1,2-dioxetane substrate ofthis invention being used. As avidin or streptavidin bind avidly tobiotin, biotinylated DNA fragments will now be tagged with an enzyme.For example, when the chemiluminscent substrate is disodium3-(4-methoxyspiro 1,2-dioxetane-3,2'-(5'-chloro) tricyclo 3.3.1.1³,7!decan!-4-yl)phenyl phosphate dioxetane salt (Cl-AMPPD), avidin orstreptavidin will be conjugated to a phosphatase. Similarly, when thechemiluminescent substrate is disodium 3-(4-methoxyspiro1,2-dioxetane-3,2'-(5'-chloro)tricyclo 3.3.1.1³,7 !decan!-4-yl)phenylβ-D-galactopyranose (Cl-AMPGD), avidin or streptavidin are conjugatedwith β-galactosidase.

Following generation of luminescence by contacting the complex of DNAfragment-biotin-avidin (or streptavidin)-enzyme with the appropriate1,2-dioxetane at alkaline pH values, e.g., above about pH 8.5, DNAfragments are visualized on light-sensitive film, e.g., X-ray or instantfilm, or in a photoelectric luminometer instrument.

The detection method outlined above can also be applied to the genomicDNA sequencing protocol of Church et al. Church, G. M., et al., Proc.Nat. Acad. Sci. (USA), 81:1991 (1984)!. After transferring chemicallycleaved and electrophoretically separated DNA Maxam, A. M. et al., Proc.Nat. Acad. Sci. (USA), 74:560 (1977)! to a membrane, preferably a nylonmembrane, and cross-linking the ladders to the membrane by UV light,specific DNA sequences may be detected by sequential addition of:biotinylated oligonucleotides as hybridization probes; avidin orstreptavidin covalently linked to an enzyme specific for anenzyme-cleavable chemiluminescent 1,2-dioxetane of this invention; and,the appropriate 1,2-dioxetane. Images of sequence ladders (produced byPAGE) may be obtained as described above.

Serial reprobing of sequence ladders can be accomplished by firststripping the hybridized probe and chemiluminescent material from amembrane by contacting the membrane with a heated solution of detergent,e.g., from about 0.5 to about 5% sodium dodecylsulfate (SDS) in water atfrom about 80° C. to about 90° C., cooling to from about 50° C. to about70° C., hybridizing the now-naked DNA fragments with anotherbiotinylated oligonucleotide probe to generate a different sequence,then generating an imaging chemiluminescence as described above.

Similar detection methods can be applied to RNA fragments generated byRNA sequencing methods.

In order that those skilled in the art can more fully understand thisinvention, the following examples are set forth. These examples aregiven solely for purposes of illustration, and should not be consideredas expressing limitations unless so set forth in the appended claims.All parts and percentages are weight by volume, except TLC solventmixtures, which are volume by volume, or unless otherwise stated.

The ¹ H NMR data given in certain of these examples for enol etherintermediates uses the prime symbol (') to designate aromatic protons,while non-primed numbers refer, in all cases, to substituentadamant-2'-ylidene ring positions, thus: ##STR19##

EXAMPLE I

Two hundred grams (1.64 mol) of 3-hydroxybenzaldehyde and 270 ml. (1.93mol) of triethylamine were charged to a flask containing one liter ofmethylene chloride in an ice bath. The resulting brown solution wasmechanically stirred, and 212 ml (1.72 mol) of trimethylacetyl chloridewas added in a thin stream from an addition funnel over a 15 minuteperiod. The resulting slurry was stirred for an additional 15 minutes,the ice bath was removed, and the reaction was then allowed to proceedfor an additional two hours. TLC (K5F; 25% acetone-hexanes) showed theabsence of starting material and a single higher R_(f) product. Thereaction mixture was transferred to a separatory funnel and admixed with250 ml of 1M hydrochloric acid. The organic phase was then extractedwith water (2×400 ml) and finally dried over sodium sulfate. The driedsolution was passed through a silica gel plug, rotary evaporated, andthen pumped under vacuum (1.0 mm Hg) to give 348 g of a greenish-brownoil: 3-pivaloyloxybenzaldehyde, which was held under an argonatmosphere.

EXAMPLE II

Four hundred mg of p-toluenesulfonic acid dissolved in 25 ml of methanolwas added, with stirring, to the 3-pivaloyloxybenzaldehyde of Example I.Trimethylorthoformate (224 ml; 2.05 mol) was then added dropwise. Theminor exotherm that resulted was allowed to proceed unchecked while themixture was stirred for 1 hour. One half gram of sodium bicarbonate wasadded, and the flask was placed on the rotary evaporator (bathtemperature 40° C.) to remove all volatiles. The resulting oil waspassed through a short silica gel column under nitrogen pressure to givean orange-brown oil which was pumped under vacuum (1.0 mm Hg) withstirring to yield 426 g of crude 3-pivaloyloxybenzaldehyde dimethylacetal. Infrared analysis revealed no aldehyde carbonyl absorption (1695cm⁻¹).

EXAMPLE III

The crude 3-pivaloyloxybenzaldehyde dimethyl acetal of Example II wasdissolved in one liter of methylene chloride, freshly distilled from P₂O₅, under an argon atmosphere in a 3 liter flask. Then, 347 ml (2.03mol) of triethyl phosphite was added all at once. The flask was fittedwith a septum inlet adapter and cooled in a dry ice-acetone bath underslight argon pressure. Boron trifluoride etherate (249 ml; 2.03 mol) wasadded in several portions by syringe, with vigorous stirring. Theresulting reaction mixture was stirred at -55° C. for two hours, thenstored in a freezer at -20° C. overnight.

Next, the flask was warmed to room temperature and its contents stirredfor 4 hours. The orange-brown solution was then poured carefully into avigorously stirred slurry of 170 g of sodium bicarbonate in 800 ml ofwater at a rate such that vigorous foaming was avoided. After vigorouslystirring the biphasic mixture for one hour the layers were thenseparated in a separatory funnel and the aqueous layer was againextracted with methylene chloride (2×250 ml). The combined organicextracts were dried over sodium sulfate, concentrated, and vacuumdistilled to yield 535 g of diethyl1-methoxy-1-(3-pivaloyloxyphenyl)methane phosphonate as a clear, lightyellow oil (b.p. 158°-161° C. at 0.25 mm Hg). This represented a 91%yield for the overall procedure of Examples I-III.

¹ HNMR (400 MHz; CDCC₃): δ1.21 and 1.25 (6H, two t, 7 Hz, OCH₂ CH₃);3.37 (3H, s, ArCHOCH₃); 3.80 (3H, s, ArOCH₃); 3.90-4.10 (4H, m, OCH₂CH₃); 4.46 (1H, d, 15.6 Hz, ArCHPO); 6.85 (1H, m); 7.00 (2H, m); 7.26(1H, m).

IR (neat): 2974, 1596, 1582, 1480, 1255 (P═O), 1098, 1050, 1020, 965cm⁻¹.

EXAMPLE IV

A solution of diisopropylamine (11.6 ml, 82.8 mmol) in 75 ml oftetrahydrofuran was cooled to -78° C. in a dry ice--acetone bath underan argon atmosphere. Thirty ml of a 2.5M solution of n-butyllithium inhexanes (Aldrich, 75.0 mmol) was added by syringe and, after stirringthe resulting lithium diisopropylamide solution for 20 minutes, 13.47 g(37.6 mmol) of diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methanephosphonate in 25 ml of tetrahydrofuran was added dropwise from anaddition funnel over a 5 minute period. The resulting red solution wasstirred at low temperature for another 30 minutes to ensure completeformation of the phosphonate carbanion.

A solution of 4.99 g (30.1 mmol) of 5-hydroxyadamantan-2-one in 25 ml oftetrahydrofuran was then added dropwise. The resulting slightly cloudymixture was stirred for 5 minutes at -78° C. and then slowly warmed toroom temperature over 40 minutes. The solution, now orange in color, wasrefluxed for 90 minutes, cooled, diluted with 200 ml of a saturatedsodium bicarbonate solution, and extracted with ethyl acetate (3×75 ml).The combined organic extracts were washed with a saturated sodiumchloride solution, quickly dried over sodium sulfate and concentrated togive 12.49 g of an orange gum, a mixture of phenolic enol ether and itspivaloate ester.

¹ HNMR (Pivaloate ester, 400 MHz, in CDCl₃ : δ7.33 (1H, m, H-5'), 7.12(1H, d, J=7.7 Hz, ArH), 6.95-7.02 (2H, m, ArH), 3.43 (1H, br. 8, H-1),3.28 (3H, s, OMe), 2.79 (1H, br. s, H-3), 2.23 (1H, br. s, H-7),1.59-1.87 (11H, m), 1.34 (9H, s, COC(CH₃)₃).

IR (in CHCl₃): 3590, 3442 (OH), 2924, 2848, 1742 (ester C═O), 1665,1602, 1578, 1426, 1274, 1152, 1118, 918 cm⁻¹.

This mixture was taken up in 100 ml of methanol and refluxed for 3.5hours in the presence of 10.7 g of anhydrous potassium carbonate. Themethanol was then stripped off and the residue partitioned between waterand ethyl acetate. The organic layer was washed with a saturated sodiumchloride solution and rotary evaporated to a residue which wasrecrystallized from chloroform-petroleum ether to give 6.5 g of3-(methoxy-5-hydroxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenol asan off-white solid (m.p. 171°-172° C.). An additional 0.80 g wasobtained from the mother liquor, giving an overall yield of phenolicenol ether of 79%, based on 5-hydroxyadamantan-2-one.

¹ HNMR (400 MHz, in CDCl₃): δ7.18 (1H, dd, J=8.4, 7.7 Hz, H-5'),6.75-6.88 (3H, m, ArH), 6.36 (1H, br. s, ArOH), 3.41 (1H, br. s, H-1),3.28 (3H, s, OMe), 2.79 (1H, br. s, H-3), 2.22 (1H, br. s, H-7),1.56-1.98 (11H, m).

IR (in CHCl₃): 3586, 3320 (OH), 3000, 2920, 2844, 1665, 1590, 1578,1445, 1296, 1092, 885 cm⁻¹.

HRMS calc. for C₁₈ H₂₂ O₃ (M⁺) 286.1573, found 286.1569.

EXAMPLE V

A solution of 5.04 g (17.6 mmol) of 3-(methoxy-5-hydroxytricyclo3.3.1.1³,7 !dec-2-ylidenemethyl)phenol in 35 ml of tetrahydrofuran,prepared under argon, was admixed with 3.4 ml (24.6 mmol) oftriethylamine and then cooled to 0° C. in an ice bath.2-Chloro-2-oxo-1,3,2-dioxaphospholane (1.95 ml, 21.1 mmol) was addeddropwise with stirring. After 5 minutes the ice bath was removed, andstirring was continued for 45 minutes at room temperature. The reactionmixture was diluted with 30 ml of anhydrous diethyl ether and filteredunder argon to exclude moisture. The triethylamine hydrochloride wasthen washed further with 20 ml of diethyl ether and the filtrateconcentrated on the rotary evaporator to give the phosphate triester asa viscous, light orange oil.

The triester, dissolved in 30 ml of molecular sieve-drieddimethylformamide under argon, was reacted for 3.5 hours at roomtemperature with 1.02 g (20.8 mmol) of dry sodium cyanide, added all atonce with stirring. The solvent was then removed under vacuum (1.0 mmHg) with heating to 50° C. A sample of the resulting orange-brownresidue, when dissolved in water and subjected to reverse phaseanalytical chromatography 0.1% sodium bicarbonate (water)--acetonitrilegradient! on a PLRP polystyrene column (Polymer Laboratories), evidencedcomplete reaction to the intermediate cyanoethyl phosphate diestersodium salt.

The residue was then taken up in 35 ml of methanol and treated dropwisewith 4.85 ml of a 4.37M solution (21.2 mmol) of sodium methoxide inmethanol, with stirring, for 30 minutes at room temperature. Reversephase analytical HPLC showed β-elimination to the phosphate monoester tobe complete. The solvent was removed and the residue triturated with 10%water/acetone to give a gummy solid. Further trituration with 3%water/acetone gave a hard, off-white solid which was filtered and driedunder vacuum (1.0 mm Hg) to give 8.35 g of crude disodium3-(methoxy-5-hydroxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl) phenylphosphate, contaminated with inorganic salts.

Reverse phase preparative HPLC using a water-acetonitrile gradient on aPLRP polystyrene column (Polymer Laboratories) and lyophilization of theappropriate fractions gave 5.2 g (72%) of purified compound as a white,granular solid, softening at 120° C. and melting to a light brown gum at163°-168° C.

¹ HNMR (400 MHz, in D₂ O): δ7.16 (1H, dd, J=8.3, 7.4 Hz, H-5'), 7.05(1H, d, J=8.3 Hz, ArH), 6.93 (1H, br. s, H-2'), 6.86 (1H, d, J=7.4 Hz,ArH), 3.2 (3H, s, OMe), 3.17 (1H, br. s, H-1), 2.61 (1H, br. s, H-3),2.06 (1H, br. s, H-7) 1.42-1.73 (10H, m).

Elemental analysis showed that the phosphate salt exists as a dihydrate.Anal. Calc. for C₁₈ H₂₁ Na₂ O₆ P.2H₂ O:C, 48.44, H, 5.65, P, 6.94.Found: C, 48.37, H, 5.90, P, 6.87.

EXAMPLE VI

A solution of 0.8 g of disodium 3-(methoxy-5-hydroxytricyclo 3.3.1.1³,7!dec-2-ylidenemethyl) phenyl phosphate in 96 ml of 25% anhydrousmethanol/chloroform containing 5.35×10⁻⁵ M methylene blue sensitizingdye was divided among three glass tubes. Each tube was then cooled to 5°C. in a water bath and saturated with oxygen by passing a stream of thegas through the solution. After 5 minutes, and while continuing tobubble oxygen through the solution, the tubes were irradiated with lightfrom a cooled, 250 watt high pressure sodium vapor lamp whilemaintaining the temperature at 5° C. A 5 mil thick piece of Kaptonpolyimide film (duPont), placed between the sodium vapor lamp and thetubes, filtered out unwanted UV radiation. After 20 minutes ofirradiation the solutions had turned pink. Reverse phase analytical HPLC0.1% sodium bicarbonate (water)--acetonitrile gradient! on a PLRPpolystyrene column (Polymer Laboratories) showed two product peaks: anearly eluting, broadened peak (retention time=3.79 minutes) and a sharp,later eluting peak (retention time=5.77 minutes). The ratio of earlyeluting product (A) to later eluting product (B) was 1.3:1 in eachtube's solution.

The solutions were combined and the solvent removed under vacuum (25.0mm Hg) on an ice bath. The residue was then dissolved in 70 ml of waterand filtered through a 0.45 μm nylon membrane. Reverse phase preparativeHPLC (water-acetonitrile gradient) easily separated two products.

Combination of the appropriate preparative HPLC fractions, followed byanalytical HPLC, showed them to be homogeneous. Lyophilization gave 0.32g of one product (A) and 0.26 g of another (B), which were shown by ¹HNMR to be isomeric (syn and anti) 1,2-dioxetanes, i.e., syn-disodium3-(4-methoxyspiro- 1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo- 3.3.1.1³,7!decan!-4-yl)phenyl phosphate: ##STR20## and its anti-isomer(anti-disodium 3-(4-methoxyspiro-1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo 3.3.1.1³,7 !decan!-4-yl)phenylphosphate): ##STR21##

Each of these isomers produced chemiluminescence, with unique light vs.time and noise profiles, upon cleavage at pH 10 in aqueous buffer mediumwith alkaline phosphatase.

¹ HNMR (A isomer, 400 MHz, in D₂ O): δ6.98-7.6 (4H, m, ArH), 3.11 (3H,s, OMe), 2.97 (1H, br. s, H-1), 2.34 (1H, br. s, H-3), 1.79 (1H, br. s,H-7), 1.3-1.68 (8H, m), 1.01 (1H, d, J=13.5 Hz), 0.8 (1H, d, J=13 Hz).

¹ HNMR (B isomer, 400 MHz, in D₂ O): δ6.94-7.62 (4H, m, ArH), 3.09 (3H,s, OMe), 2.91 (1H, br. s, H-1), 2.27 (1H, br. s, H-3), 1.84 (1H, br. s,H-7), 1.21-1.75 (8H, m), 1.02 (1H, d, J=12.8 Hz), 0.87 (1H, d, J=12.8Hz).

Elemental analysis showed that isomer B exists as a dihydrate. Anal.Calc. for C₁₈ H₂₁ Na₂ O₈ P.2H₂ O (isomer B): C, 45.2, H, 5.27, P, 6.47.Found: C, 45.53, H, 5.35, P, 6.11.

It was not possible to specifically designate which of the two isomersobtained was the syn-isomer and which was the anti-isomer on the basisof ¹ HNMR data.

EXAMPLE VII

Following in general the procedure of Example IV above, a solution of5.35 ml (38.2 mmol) of diisopropylamine in 35 ml of tetrahydrofuran wascooled to -78° C. in an ice bath under an argon atmosphere. Fourteen mlof a 2.5M solution of n-butyllithium in hexanes (35.0 mmol) was added bysyringe and, after stirring for 20 minutes, 10.86 g (30.3 mmol) ofdiethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methane phosphonate in 30 ml oftetrahydrofuran was added dropwise over a 10 minute period. Theresulting orange solution was stirred at low temperature for 1 hour,then admixed with a solution of 5.21 g (22.75 mmol) of5-bromoadamantan-2-one in 20 ml of tetrahydrofuran over a 7 minuteperiod, with stirring. Stirring was continued for an additional 10minutes, at which point the cold bath was removed and the reactionmixture was allowed to warm slowly to room temperature over a one hourperiod.

The solution was then refluxed for another hour, cooled, diluted with100 ml of hexanes, and poured into a separatory funnel containingsaturated aqueous sodium bicarbonate solution and extracted with 10%ethyl acetate in hexanes (3×50 ml). The combined organic extracts werewashed with an aqueous 15% sodium chloride solution, quickly dried oversodium sulfate, and concentrated to give 11.62 g of a light orangeviscous oil. Plug filtration on a short silica gel column, eluting with10% ethyl acetate in n-hexanes, gave 11.0 g of a yellowish-green gum.

¹ HNMR (Pivaloyl ester, 400 MHz, in CDCl₃): δ7.34 (1H, t, J=7.8 Hz,H-5'), 7.1 (1H, d, J=7.7 Hz, ArH), 6.95-7.02 (2H, m, ArH), 3.39 (1H, br.s, H-1), 3.28 (3H, s, OMe), 2.74 (1H, br. s, H-3), 2.32-2.51 (6H, m,H-4, H-6, H-9), 2.17 (1H, br. s, H-7), 1.67-1.92 (4H, m, H-8, H-10),1.34 (9H, s, COC(CH₃)₃).

IR (neat): 2924, 2850, 1745 (ester C═O), 1654, 1602, 1578, 1478, 1274,1110, 1018, 808, 758 cm⁻¹.

This gum was taken up in 30 ml of methanol and refluxed for 2 hours with2.4 g of anhydrous potassium carbonate. The methanol was then removedand the residue partitioned between water and 30% ethyl acetate inhexanes. The organic layer was washed with aqueous saturated sodiumchloride solution, dried over sodium sulfate and concentrated to give9.32 g of a yellow gum. This gum was flash chromatographed on silica gelto give 7.06 g (88% yield based on 5-bromoadamantan-2-one) of a slightlyyellow gum that could not be crystallized. IR and NMR indicated,however, that the compound obtained, 3-(methoxy-5-bromotricyclo3.3.1.1³,7 !dec-2-ylidenemethyl)phenol, was pure enough to be used forthe ensuing phosphorylation reaction.

A sample of the pure phenolic compound was obtained from3-(methoxy-5-bromotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl) phenylacetate, obtained as follows: The impure phenolic compound (5.57 g;15.95 mmol) was dissolved in 30 ml of molecular sieve-dried pyridineunder an argon atmosphere. Fifty mg of 4-dimethylaminopyridine was addedas catalyst. Next, 1.8 ml (19.15 mmol) of acetic anhydride was added bysyringe and the reaction mixture was stirred at room temperature for 3hours.

The reaction mixture was then transferred to a separatory funnelcontaining 250 ml of an aqueous saturated sodium bicarbonate solution,then extracted with 10% ethyl acetate in hexanes (2×100 ml). Thecombined extracts were washed several times with water, then quicklydried over sodium sulfate. The dried solution was concentrated on arotary evaporator, and the residue was recrystallized from n-hexanescontaining a few drops of ethyl acetate. The thus-obtained off-whitesolid was again recrystallized to give 5.21 g (83.5% yield) of theacetate, m.p. 108°-110° C.

HRMS calc. for C₂₀ H₂₃ BrO₃ (M⁺) 390.0833, found 390.0831.

¹ HNMR (400 MHz, in CDCl₃): δ7.35 (1H, dd, J=8, 7.7 Hz, H-5'), 7.13 (1H,dd, J=7.7, 1 Hz, ArH), 7.03 (1H, dd, J=8, 1 Hz, ArH), 6.99 (1H, d, 1 Hz,H-2'), 3.39 (1H, br. s, H-1), 3.27 (3H, s, OMe), 2.75 (1H, br. s, H-3),2.32-2.51 (6H, m, H-4, H-6, H-9), 2.29 (3H, s, OAc), 2.18 (1H, br. s,H-7), 1.69-1.92 (4H, m, H-8, H-10).

IR (in CHCl₃): 3000, 2930, 2850, 1760 (ester C═O), 1660, 1602, 1577,1368, 1192, 1095, 1070, 1016, 804 cm⁻¹.

Treatment of the recrystallized acetate with potassium carbonate inmethanol for 15 hours at room temperature gave3-(methoxy-5-bromotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl!phenol, m.p.42°-45° C., as a white, crispy foam that could not be recrystallizedfurther.

¹ HNMR (400 MHz, in CDCl₃): δ7.21 (1H, t, J=7.2 Hz, H-5'), 6.73-6.85(3H, m, ArH), 5.18 (1H, s, ArOH), 3.38 (1H, br. s, H-1), 3.28 (3H, s,OMe), 2.74 (1H, br. s, H-3), 2.3-2.52 (6H, m, H-4, H-6, H-9), 2.17 (1H,br. s, H-7), 1.68-1.92 (4H, m, H-8, H-10).

IR (in CHCl₃): 3584, 3320 (OH), 2925, 2850, 1665, 1588, 1578, 1445,1435, 1092, 1080, 1015, 880, 808 cm⁻¹.

EXAMPLE VIII

Again following in general the procedure of Example IV above, a solutionof 2.97 ml (21.3 mmol) of diisopropylamine in 21 ml of tetrahydrofuranwas cooled to -78° C. in a dry ice-acetone bath under an argonatmosphere, admixed with 8.5 ml of a 2.5M solution of n-butyllithium inhexanes (21.3 mmol), added dropwise by syringe, and stirred for 20minutes. Diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methane phosphonate(7.26 g; 20.3 mmol) in 20 ml of tetrahydrofuran was then added dropwiseby syringe over a 10 minute period, and the solution was stirred at lowtemperature for 1 hour.

A solution of 2.79 g (15.2 mmol) of 5-chloroadamantan-2-one in 15 ml oftetrahydrofuran was added over a 5 minute period and, after stirring atlow temperature for 10 minutes, the cold bath was removed and themixture warmed to room temperature. The mixture was then refluxed for1.5 hours, cooled, diluted with 50 ml of n-hexanes, and poured into aseparatory funnel containing 150 ml of an aqueous saturated sodiumbicarbonate solution. Extraction with 5% ethyl acetate-n-hexanes wasfollowed by drying the combined organic fractions, concentrating themand pumping them under vacuum (1.0 mm Hg) to yield a residue which, whenchromatographed on silica gel, gave 5.15 g of the higher R_(f)3-(methoxy-5-chlorotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenyltrimethylacetate as a colorless oil (structure confirmed by IR and NMR),and 0.865 g of a later-eluting material composed predominantly of thecorresponding phenol. This latter fraction, when reacylated withpivaloyl chloride and triethylamine in methylene chloride (see Example Iabove), followed by chromatography, gave an additional 0.35 g of thepivaloyl ester (93% total yield based on 5-chloroadamantan-2-one).

¹ HNMR (Pivaloyl ester, 400 MHz, in CDCl₃): δ7.36 (1H, t, J=7.8 Hz,H-5'), 7.13 (1H, d, J=7.7 Hz, ArH), 6.98-7.04 (2H, m, ArH), 3.45 (1H,br. s, H-1), 3.3 (3H, s, OMe), 2.8 (1H, br. s, H-3), 2.13-2.32 (7H, m,H-4, H-6, H-7, H-9), 1.65-1.9 (4H, m, H-8, H-10), 1.36 (9H, s,COC(CH₃)₃).

IR (neat): 2932, 2835, 1750 (ester C═O), 1664, 1602, 1578, 1478, 1274,1112, 1022, 827, 758 cm⁻¹.

The combined pivaloyl ester fractions (5.5 g, 14.1 mmol) were taken upin 40 ml of methanol and refluxed with 5.37 g of anhydrous potassiumcarbonate for 40 minutes. The residue remaining after the methanol wasstripped off was partitioned between water and 30% ethylacetate-hexanes, and the organic fractions were concentrated and plugfiltered on a short silica gel column to give 4.07 g (88% yield based on5-chloroadamantan-2-one) of 3-(methoxy-5-chlorotricyclo 3.3.1.1³,7!dec-2-ylidenemethyl) phenol as a crispy white foam which becameslightly tacky upon exposure to air.

HRMS calc. for C₁₈ H₂₁ ClO₂ (M⁺) 304.1227, found 304.1230.

¹ HNMR (400 MHz, in CDCl₃): δ7.23 (1H, dd, J=7.7, 7.6 Hz, H-5'), 6.85(1H, d, J=7.6 Hz, ArH), 6.77-6.83 (2H, m, ArH), 3.44 (1H, br. s, H-1),3.31 (3H, s, OMe), 2.8 (1H, br. s, H-3), 2.1-2.31 (7H, m, H-4, H-6, H-7,H-9), 1.65-1.89 (4H, m, H-8, H-10).

IR (in CHCl₃): 3590, 3330 (OH), 2930, 2855, 1655, 1590, 1580, 1440,1295, 1094, 1080, 1022, 880, 826 cm⁻¹.

EXAMPLE IX

3-(Methoxy-5-bromotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenol (1.49g, 4.26 mmol) was dissolved in 8.5 ml anhydrous ethylene glycol and thenplaced, together with 0.28 g of potassium carbonate, in a sealed glasstube. The tube was heated in an oil bath at 110° C. for seven hours. Thecontents of the tube were then concentrated in vacuo (1.0 mm Hg) with )heating. The residue was partitioned between saturated sodium chloridesolution and ethyl acetate. The organic fraction was then stripped andchromatographed on a short silica gel column to furnish 1.31 g (92%yield based on the phenol starting material) of3-(methoxy-5-(2-hydroxy)ethoxytricyclo 3.3.1.1³,7!dec-2-ylidenemethyl)phenol as an off-white foam.

¹ HNMR (400 MHz CDCl₃): δ7.19 (1H, t, J=7.6 Hz, H-5'), 6.83 (1H, d,J=7.6 Hz, ArH), 6.73-6.80 (2H, m, ArH), 5.83 (1H, s, ArOH), 3.67 (2H, m,OCH₂ CH₂ OH), 3.51 (2H, t, J=4.6 Hz, OCH₂ CH₂ OH), 3.44 (1H, br. s,H-1), 3.28 (3H, s, OMe), 2.81 (1H, br. s, H-3), 2.24 (1H, br. s, H-7),1.55-1.90 (10H, m).

IR (CHCl₃): 3580, 3320 (OH), 2929, 2842, 1664, 1586, 1575, 1440, 1092,1078, 885 cm⁻¹.

EXAMPLE X

3-(Methoxy-5-chlorotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenol (1.23g, 4.0 mmol) was phosphorylated in the manner described in Example Vabove with one exception--ammonia in methanol was used forβ-elimination. The crude ammonium sodium salt obtained was trituratedwith acetone, then pumped in vacuo (1.0 mm Hg) to give 1.2 g of anoff-white solid. Reverse phase analytical HPLC showed that thephosphorylated enol ether product thus obtained was pure enough fordirect photooxygenation to the corresponding 1,2-dioxetane.

The 3-(methoxy-5-chlorotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenylphosphate salt (0.65 g) was dissolved in 100 ml of anhydrous 10%methanol/chloroform which also contained 5.35×10⁻⁵ M methylene blue as asensitizing dye. The resulting solution was divided among three glasstubes and irradiated as described in Example VI above. Work-up, in thiscase, involved dissolution of the pumped residue in 70 ml of watercontaining 258 mg of sodium bicarbonate. Upon carrying out reverse phasepreparative HPLC, the syn- and anti-isomers were collected together,excluding impurities. Analytical HPLC 0.1% NaHCO₃ (H₂ O)-acetonitrilegradient! showed two product peaks (retention times of 8.01 and 8.32minutes). The area percent ratio (270 nm) of the early eluting isomer tothe later eluting isomer was found to be 0.4:1. ¹ H NMR confirmed thatthe lyophilized white solid obtained was a mixture of syn- andanti-disodium 3-(4-methoxyspiro- 1,2-dioxetane-3,2'-(5'-chloro)tricyclo3.3.1.1³,7 !decan!-4-yl)phenyl phosphate.

Elemental analysis indicated that the product exists in the form of adihydrate. Anal. Calc. for C₁₈ H₂₀ ClNa₂ O₇ P.2H₂ O: C, 43.52; H, 4.87;Cl, 7.14. Found:C, 43.23; H, 4.99; Cl, 7.65.

¹ HNMR(400 MHz, in D₂ O, two isomers): δ6.97-7.68(4H, m, ArH), 3.08 and3.09 (3H, 2s, OMe), 2.95 (1H, br.s, H-1), 0.76-2.35 (12H, m).

EXAMPLE XI

3-(Methoxy-5-bromotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenol wasphosphorylated and then photooxygenated as described for its 5-chloroanalog in Example X above. Work-up in 0.3% (w/v) aqueous sodiumbicarbonate solution gave a filtered, aqueous solution of the crude1,2-dioxetane phosphate salt, which was then subjected to reverse phasepreparative HPLC (water-acetonitrile gradient) to give the syn- andanti-isomers, collected together, for lyophilization. When thelyophilized product, a white, fluffy solid, was subjected to analyticalHPLC, two peaks were obtained with retention times of 8.52 and 8.94minutes. The area percent ratio (270 nm) of the early eluting isomer tothe later eluting isomer was found to be 0.5:1. ¹ HNMR confirmed thatthe product was a mixture of syn- and anti-disodium 3-(4-methoxyspiro-1,2-dioxetane-3,2'-(5'-bromo)tricyclo 3.3.1.1³,7 !decan!-4-yl)phenylphosphate.

¹ HNMR (400 MHz, in D₂ O, two isomers): δ6.99-7.52(4H, m, ArH), 3.07 and3.09 (3H, 2s, OMe), 2.91 (1H, br.s, H-1), 0.82-2.32 (12H, m).

EXAMPLE XII

Immunoassays for TSH were conducted on a series of TSH standards using aHybritech Tandem-E TSH kit (Hybritech, Inc., San Diego, Calif.)according to the manufacturer's instructions included with the kit,except that upon completion of the anti-TSH-alkaline phosphataseconjugate incubation step and wash, the plastic beads were additionallywashed with 0.1M diethanolamine, 1 mM magnesium chloride, 0.02% sodiumazide buffer, pH 10.0, and then briefly stored in 200 μl of the samebuffer.

Chemiluminescent signals from anti-TSH-alkaline phosphatase conjugatebound to the surface of the beads were initiated by adding to the tubescontaining beads 300 μl of 0.67 mM buffer solutions containing,respectively, disodium3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane("AMPPD"), disodium 3-(4-methoxyspiro-1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo 3.3.1.1³,7 !decan!-4-yl)phenylphosphate (A isomer; "A-OH-AMPPD"), the corresponding disodium B-isomer("B-OH-AMPPD"), and disodium 3-(4-methoxyspiro-1,2-dioxetane-3,2'-(5'-chloro)tricyclo 3.3.1.1³,7 !decan!-4-yl)phenylphosphate ("Cl-AMPPD"), in 0.1M diethanolamine, 1 mM magnesium chloride,0.02% sodium azide, pH 10.0. The intensity of light emission wassubsequently recorded at 7, 13, 19, 25, 31, 40, 50 and 60 minutes afterthe substrate addition, as a 5 second integral at room temperature(about 25° C.), using a Berthold LB952T Luminometer (BertholdInstrument, Wildbad, Federal Republic of Germany).

TSH, RLU v. TSH for each of AMPPD, Br-AMPPD, B-OH-AMPPD, A-OH-AMPPD andCl-AMPPD is shown in FIGS. 1, 2, 3, 4 and 5, respectively.

EXAMPLE XIII

A comparison of the total luminescence emission from AMPPD and from thecorresponding 1,2-dioxetanes whose adamant-2'-ylidene groups aremonosubstituted with hydroxy (A and B isomers), chloro and bromo groupswas made by carrying out total dephosphorylation experiments on each ofthese compounds.

An aqueous solution of the 1,2-dioxetane (4.0 mM) in 0.05M sodiumcarbonate/sodium bicarbonate containing 1 mM magnesium chloride wasprepared and then equilibrated at 30° C. Ten μl of a 7.64×10M aqueoussolution of alkaline phosphatase (calf intestine; Biozyme) was thenadded, and the chemiluminescence from the resulting solution wasrecorded using a Turner Model 20E luminometer (Turner Instruments;Sunnyvale, Calif.).

The rates of chemiluminescence decay for each of the five compounds inquestion, expressed in relative light units (RLU's) per minute, aregiven in the following table.

                  TABLE I                                                         ______________________________________                                                   Decay Rate (RW's)                                                                           Total Decay                                          1.2-Dioxetane                                                                              I        II    III    Time (min).                                ______________________________________                                        AMPPD        0.1      0.3   0.8    60                                         OH-Adamant-2'-                                                                             1.3      --    --     9                                          ylidene (A isomer)                                                            OH-Adamant-2'-                                                                             1.5      --    --     10                                         ylidene (B isomer)                                                            Cl-Adamant-2'-                                                                             1.2      5.5   --     15                                         ylidene                                                                       Br-Adamant-2'-                                                                             1.2      0.8   --     40                                         ylidene                                                                       ______________________________________                                    

These total chemiluminescence emissions are depicted graphically inFIGS. 6, 7, 8, 9 and 10, respectively.

EXAMPLE XIV

A strip of neutral BIODYNE A nylon membrane (Pall Corporation, GlenCove, N.Y.) was dotted twice (side-by-side) with the followingconcentrations of biotinylated pBr 322 35-mer oligonucleotide probe(Synthetic Genetics, San Diego, Calif.):

    ______________________________________                                                      DNA                                                             Pair of Dots No.                                                                            Concentration (picograms)                                       ______________________________________                                        1             100.000                                                         2             50.000                                                          3             25.000                                                          4             12.500                                                          5             6.250                                                           6             3.125                                                           7             1.563                                                           8             0.781                                                           9             0.391                                                           10            blank.sup.1/                                                    ______________________________________                                         .sup.1/ Single stranded, unlabeled DNA, 1 ng.                            

Next, the membrane was blocked in 0.2% casein/0.1% Tween 20 detergent inaqueous phosphate buffered saline solution (PBS) for 1 hour, followingwhich 1/5000 diluted avidin-alkaline phosphatase conjugate (Sigma, Inc.,St. Louis, Mo.) in 0.2% casein in PBS was added. The membrane was thenincubated for 30 minutes, washed twice (for 5 minutes each time) in 0.2%casein/0.1% Tween 20 detergent in PBS, washed four times (for 5 minuteseach time) in 0.3% Tween 20 detergent in PBS, and twice (for 5 minuteseach time) in aqueous 0.1M diethanolamine containing 1 mM magnesiumchloride, pH 10.0.

The membrane was then cut up the middle to give two strips, each bearingone set of dots. One of the strips was incubated for 5 minutes inaqueous AMPPD solution (0.25 mM in 0.1M diethanolamine containing 1 mMmagnesium chloride, pH 10), the other for 5 minutes in the correspondingchloroadamant-2'-ylidene compound (0.25 mM in the same buffer). The twostrips were then placed in camera luminometers and exposed on PolaroidType 612 instant black and white film. The improved chemiluminescenceintensity obtained using the chloro compound, as compared to AMPPDitself, can be seen by comparing column 2 (Cl-AMPPD) to column 1 AMPPD)in FIG. 11.

EXAMPLE XV

pBr 322 plasmid (4700 bp) was subjected to a nick translation processusing a Trans-Light kit (Tropix, Inc., Bedford, Mass.) to generate amixture of biotinylated single stranded polynucleotides of 200-2000 bpsin length.

This mixture was dotted onto a dry BIODYNE A membrane as five parallelcolumns of dots of the following concentrations:

    ______________________________________                                                      DNA                                                             Row of Dots No.                                                                             Concentration (picograms)                                       ______________________________________                                        1             20.000                                                          2             10.000                                                          3             5.000                                                           4             2.500                                                           5             1.250                                                           6             0.625                                                           7             0.313                                                           8             0.156                                                           9             0.078                                                           10            0.039                                                           ______________________________________                                    

The membrane was subjected to ultraviolet irradiation (UVP MineralLight; UVP, San Gabriel, Calif.) for 3 minutes to fix the DNA to thesurface of the membrane, then air dried. Next, the membrane was blockedin 0.2% casein/0.1% Tween 20 detergent in PBS for 1 hour, followingwhich 1/5000 diluted avidin-alkaline phosphatase conjugate (Tropix,Inc.) in 0.2% casein in PBS was added. The membrane was then incubatedfor 30 minutes, washed three times (for 5 minutes each time) in 0.2%casein/0.1% Tween 20 detergent in PBS and washed once for five minutesin aqueous 0.1M diethanolamine containing 1 mM magnesium chloride and0.02% sodium azide, pH 10.0 (substrate buffer).

Next, the five columns of rows of dots 1-5 were individually cut fromthe membrane ("strips 1-5"), as were the five columns of rows of dots6-10 ("strips 6-10"). Strips 1-5 were washed with substrate buffer for30 minutes. Strips 6-10 were blocked in 0.1% BDMQ in substrate bufferfor 30 minutes. Both sets of strips were then incubated for 5 minutes insubstrate buffer, then individually incubated for five minutes inaqueous solutions (0.25 mM) of 1,2-dioxetanes as indicated below:

    ______________________________________                                        Strip No.     1.2-Dioxetane                                                   ______________________________________                                        1             AMPPD                                                           2             OH-Adamant-2'-ylidene (A isomer)                                3             OH-Adamant-2'-ylidene (B isomer)                                4             Cl-Adamant-2'-ylidene                                           5             Br-Adamant-2'-ylidene                                           6             AMPPD                                                           7             OH-Adamant-2'-ylidene (A isomer)                                8             OH-Adamant-2'-ylidene (B isomer)                                9             Cl-Adamant-2'-ylidene                                           10            Br-Adamant-2'-ylidene                                           ______________________________________                                    

All strips were then placed in camera luminometers and exposed onPolaroid Type 612 instant black and white film. The improvedchemiluminescence intensity obtained using the 3-(substitutedadamant-2'-ylidene)1,2-dioxetanes, as compared to AMPPD itself, can beseen from the results shown in Table II below.

                                      TABLE II                                    __________________________________________________________________________    ALKALINE PHOSPHATASE-LABELED DNA PROBE DETECTION IN MEMBRANE                  WITH AND WITHOUT BDMQ BLOCKING STEP                                                                       Detection                                                                            (in pico-                                                       BDMQ   limit  grams DNA)                                 Membrane #                                                                          Dioxetane      Blocking Step                                                                        5. min. exp..sup.1/                                                                  1 min. exp..sup.2/                         __________________________________________________________________________    1     AMPPD          no     2.500  5.000                                      2     OH-Adamant-2'-ylidene (A isomer)                                                             no     5.000  10.000                                     3     OH-Adamant-2'-ylidene (B isomer)                                                             no     5.000  20.000                                     4     CL-Adamant-2'-ylidene                                                                        no     1.250  1.250                                      5     Br-Adamant-2'-ylidene                                                                        no     1.250  2.500                                      6     AMPPD          BDMQ   0.625  1.250                                      7     OH-Adamant-2'-ylidene (A isomer)                                                             BDMQ   0.625  2.500                                      8     OH-Adamant-2'-ylidene (B isomer)                                                             BDMQ   1.250  5.000                                      9     Cl-Adamant-2'-ylidene                                                                        BDMQ   0.313  0.313                                      10    Br-Adamant-2'-ylidene                                                                        BDMQ   0.313  0.313                                      __________________________________________________________________________     .sup.1/ Five minute exposure was performed 40 minutes after dioxetane         addition.                                                                     .sup.2/ One minute exposure was performed 77 minutes after dioxetane          addition.                                                                

EXAMPLE XVI

A strip of neutral BIODYNE A nylon membrane was dotted twice(side-by-side) with 12.5 picograms of biotinylated pBR 322 35-meroligonucleotide probe (Biogen, Inc., Cambridge, Mass.), dried, andsubjected to ultraviolet radiation from an UVP Mineral Light lamp for 5minutes to fix the DNA to the surface of the membrane. Next, themembrane was wetted with 5×SSC (0.015M sodium citrate/0.15M sodiumchloride) and blocked in 0.2% casein, 0.1% Tween 20 detergent in PBS for1 hour. The blocked membrane was then incubated with avidin-alkalinephosphatase conjugate ("Avidx" conjugate; Tropix, Inc.), diluted1-10,000 in 0.2% casein, for 30 minutes.

The membrane was then washed twice (for 5 minutes each time) withaqueous 0.2% casein/0.1% Tween 20 detergent, then twice (for 5 minuteseach time) with aqueous 0.1% Tween 20 detergent in PBS, and finallytwice (for 5 minutes each time) in aqueous 0.1M diethanolaminecontaining 1 mM magnesium chloride, pH 10 (assay buffer).

The washed membrane was cut in half to give two strips, each bearing onedot. The strips were incubated, respectively, in aqueous 0.25 mMsolutions of AMPPD and the corresponding chloroadamant-2'-ylidenecompound in 0.1M diethanolamine containing 1 mM magnesium chloride, pH10 for five minutes, then drained and sealed in plastic bags which weretaped to the window of a Turner Model 20E luminometer. Thechemiluminescence emission from each strip was integrated for 20 hours.The kinetics of the light emissions obtained are shown in FIG. 12.

EXAMPLE XVII

The enhancement of chemiluminescence emission (as compared to theemission from AMPPD) provided by the correspondinghydroxyadamant-2'-ylidene (A and B isomers) and chloroadamant-2'-ylidenecompounds, all in the further presence of the enhancer polymers listedbelow, was demonstrated in the following manner.

Four sets of three tubes each, each tube in each set containing 450 μlof a 0.4 mM aqueous solution of one of the four 1,2-dioxetanes beingcompared in 0.1M diethanolamine containing 1 mM magnesium chloride,0.02% sodium azide and 0.1% of the enhancer polymer, pH 10.0 (substratebuffer) were prepared and the background signal from each tube measuredusing a Berthold LB 952T luminometer (Berthold Instruments; Wildbad,Federal Republic of Germany).

Next, 50 μl of an aqueous solution, 2.83×10⁻¹² M, of alkalinephosphatase in 0.1M diethanolamine containing 1 mM magnesium chloride,0.02% sodium azide, pH 10.0, (final enzyme concentration 2.83×10⁻¹³ M)was added to each tube and the chemiluminescent signals were measured inthe luminometer at 5 and 20 minutes. The intensity of thechemiluminescent signals and the signal: background ratios obtained foreach of the four 1,2-dioxetanes in the presence of the enhancer polymersare shown in Table III below.

The enhancer polymers used, and the symbols for such polymers used inTable III, were the following:

                                      TABLE III                                   __________________________________________________________________________               AMPPD                                                              POLYMER                                                                              TIME                                                                              SIGNAL                                                                             S/N                                                                              SIGNAL                                                                             S/N                                                                              SIGNAL                                                                             S/N                                                                              SIGNAL                                                                             S/N                                   __________________________________________________________________________    NONE   0   172.6                                                                              1.0                                                                              87.0 1.0                                                                              126.3                                                                              1.0                                                                              85.3 1.0                                          5   3706.0                                                                             21.6                                                                             2653.6                                                                             32.9                                                                             3314.8                                                                             26.3                                                                             4063.6                                                                             47.9                                         20  5944.8                                                                             34.4                                                                             3160.8                                                                             36.3                                                                             3767.9                                                                             29.8                                                                             4460.9                                                                             52.3                                  SAPPHIRE                                                                             0   241.4                                                                              1.0                                                                              86.1 1.0                                                                              138.8                                                                              1.0                                                                              296.0                                                                              1.0                                          5   38457.8                                                                            159.3                                                                            21051.3                                                                            244.5                                                                            10908.4                                                                            78.6                                                                             51584.5                                                                            371.6                                        20  85359.8                                                                            351.6                                                                            25130.3                                                                            292.3                                                                            14625.4                                                                            105.3                                                                            70161.6                                                                            505.4                                 TMQ    0   159.9                                                                              1.0                                                                              77.9 1.0                                                                              125.7                                                                              1.0                                                                              60.3 1.0                                          5   10525.8                                                                            65.8                                                                             8853.4                                                                             113.6                                                                            6258.9                                                                             49.8                                                                             9941.0                                                                             165.0                                        20  22558.3                                                                            141.1                                                                            11658.3                                                                            149.6                                                                            8857.8                                                                             70.5                                                                             13640.5                                                                            226.4                                 S/TMQ(1:4)                                                                           0   116.3                                                                              1.0                                                                              81.2 1.0                                                                              134.0                                                                              1.0                                                                              60.0 1.0                                          5   5931.5                                                                             35.7                                                                             9294.6                                                                             114.5                                                                            4775.3                                                                             35.6                                                                             5631.2                                                                             93.9                                         20  15866.3                                                                            95.4                                                                             1201.7                                                                             148.1                                                                            7120.3                                                                             53.1                                                                             9957.4                                                                             165.0                                 S/TMQ(1:2)                                                                           0   169.8                                                                              1.0                                                                              90.6 1.0                                                                              160.3                                                                              1.0                                                                              66.8 1.0                                          5   3437.0                                                                             20.2                                                                             10193.8                                                                            112.5                                                                            3305.1                                                                             20.6                                                                             3121.4                                                                             46.7                                         20  9994.7                                                                             58.9                                                                             11365.1                                                                            158.6                                                                            4722.3                                                                             35.7                                                                             6896.9                                                                             100.2                                 DAA TMQ                                                                              0   180.0                                                                              1.0                                                                              90.5 1.0                                                                              136.0                                                                              1.0                                                                              82.6 1.0                                          5   8354.4                                                                             46.4                                                                             5583.1                                                                             61.7                                                                             5486.8                                                                             40.3                                                                             10326.7                                                                            125.1                                        20  14957.3                                                                            83.1                                                                             6309.4                                                                             69.7                                                                             8670.3                                                                             49.0                                                                             13204.2                                                                            159.9                                 DMQ TEQ                                                                              0   245.2                                                                              1.0                                                                              90.8 1.0                                                                              145.5                                                                              1.0                                                                              77.2 1.0                                          5   32820.5                                                                            133.9                                                                            17235.6                                                                            189.7                                                                            9213.8                                                                             63.3                                                                             39712.6                                                                            514.6                                        20  78977.4                                                                            322.1                                                                            20702.2                                                                            227.9                                                                            8268.0                                                                             57.0                                                                             57658.0                                                                            747.2                                 TEQ    0   217.8                                                                              1.0                                                                              348.8                                                                              1.0                                                                              142.6                                                                              1.0                                                                              67.1 1.0                                          5   25095.8                                                                            115.3                                                                            15779.8                                                                            45.24                                                                            8806.2                                                                             61.8                                                                             26551.0                                                                            397.0                                        20  50020.0                                                                            229.7                                                                            1906.9                                                                             54.7                                                                             11563.4                                                                            81.1                                                                             34467.2                                                                            513.5                                 TBQ    0   493.9                                                                              1.0                                                                              101.8                                                                              1.0                                                                              148.9                                                                              1.0                                                                              118.8                                                                              1.0                                          5   94429.4                                                                            191.2                                                                            37244.5                                                                            366.0                                                                            15508.5                                                                            97.6                                                                             148394.5                                                                           1249.6                                       20  214319.4                                                                           433.9                                                                            44569.8                                                                            430.0                                                                            20630.8                                                                            131.1                                                                            209466.8                                                                           1763.2                                MPS    0   211.8                                                                              1.0                                                                              93.6 1.0                                                                              140.3                                                                              1.0                                                                              86.0 1.0                                          5   19971.8                                                                            94.3                                                                             13165.3                                                                            140.7                                                                            10021.3                                                                            71.5                                                                             22170.5                                                                            257.8                                        20  41701.8                                                                            196.9                                                                            16159.2                                                                            172.7                                                                            10939.0                                                                            78.0                                                                             29133.9                                                                            308.5                                 BAEDM  0   217.3                                                                              1.0                                                                              95.6 1.0                                                                              161.4                                                                              1.0                                                                              88.9 1.0                                          5   9275.3                                                                             107.6                                                                            4887.3                                                                             61.6                                                                             4101.3                                                                             25.4                                                                             9066.9                                                                             102.2                                        20  26529.1                                                                            308.5                                                                            7433.5                                                                             77.8                                                                             5538.2                                                                             34.3                                                                             17185.4                                                                            193.3                                 BZ     0   149.4                                                                              1.0                                                                              79.9 1.0                                                                              119.4                                                                              1.0                                                  5   2738.0                                                                             18.3                                                                             2156.9                                                                             27.0                                                                             2246.4                                                                             18.8                                                 20  4611.5                                                                             30.9                                                                             2536.7                                                                             31.7                                                                             2669.2                                                                             22.4                                          DMEB   0   176.6                                                                              1.0                                                                              79.9 1.0                                                                              124.6                                                                              1.0                                                  5   9401.5                                                                             52.6                                                                             6160.6                                                                             77.1                                                                             4451.6                                                                             35.7                                                 20  20434.9                                                                            114.4                                                                            7835.2                                                                             98.1                                                                             6023.9                                                                             48.3                                          DME(OH)B                                                                             0   161.6                                                                              1.0                                                                              85.2 1.0                                                                              132.1                                                                              1.0                                                  5   3462.3                                                                             21.4                                                                             3931.9                                                                             46.1                                                                             2640.9                                                                             20.0                                                 20  8540.9                                                                             52.9                                                                             5300.5                                                                             66.2                                                                             3990.6                                                                             30.2                                          EMERALD                                                                              0   2902.1                                                                             1.0                3434.0                                                                             1.0                                          5   317571.6                                                                           109.4              400510.8                                                                           583.2                                        20  818775.8                                                                           282.1              662054.4                                                                           964.0                                 TBQ-FLUOR                                                                            0   4961.2                                                                             1.0                5882.7                                                                             1.0                                          5   57271.6                                                                            113.9              987572.0                                                                           839.4                                        20  1445675.6                                                                          291.4              1505971.2                                                                          1280.0                                __________________________________________________________________________    SYMBOL ENHANCER POLYMER                                                       SAPPHIRE                                                                             BDMQ                                                                   TMQ    poly(vinylbenzyltrimethylammonium chloride                             S/TMQ  styrene/TMQ copolymer                                                  DAA/TMQ                                                                              diacetone acrylamide/TMQ copolymer                                     DMQ/TEQ                                                                              poly(vinylbenzyldodecyldimethylammonium                                       chloride)/TEQ copolymer                                                TEQ    poly(vinylbenzyltriethylammonium chloride)                             TBQ    poly(vinylbenzyltributylammonium chloride)                             MPB    poly(vinylbenzyl-N-methylpiperidinium                                         chloride)                                                              BAEDM  poly vinylbenzyl(2-benzoylamino)ethyl-                                        dimethylammonium chloride!                                             BZ     benzal mordant                                                         DMEB   poly(vinylbenzyldimethylethyl-                                                ammonium chloride)                                                     DME(OH)B                                                                             poly vinylbenzyldimethyl(2-hydroxy)eth-                                       ylammonium chloride!                                                   EMERALD                                                                              sapphire and fluorescein                                               TBQ/FLUOR                                                                            TBQ and fluorescein                                                

EXAMPLE XVIII

The background signals and t1/2 parameters (as compared to those ofAMPPD) obtained for the substituted adamant-2'-ylidene compounds listedbelow in two different buffers were determined in the following manner.

Aqueous 4×10⁻⁴ M solutions of the 1,2-dioxetanes in a buffer solutionmade up of 0.05M sodium carbonate/sodium bicarbonate containing 1 mMmagnesium chloride, pH 9.5, were prepared, as were aqueous 4×10⁻⁴ Msolutions of the 1,2-dioxetanes in a buffer solution made up of 0.1Mdiethanolamine containing 1 mM magnesium chloride and 0.02% sodiumazide, pH 10.0. One ml per tube of each of these solutions was placed ina Turner Model 20E luminometer and the background signals were measured.

Next, t1/2 values were measured for each sample as follows. One hundredμl of sample dioxetane in 900 μl of one of the above-described buffersolutions was pipetted into a tube (final dioxetane concentration 4×10⁻⁵M) and equilibrated at 30° C. Ten μl of a 1-1,000 dilution of calfintestine alkaline phosphatase in the same buffer (enzyme concentration7.6×10⁻¹⁰ M) was then added, and the resulting chemiluminescentintensity was recorded, using a Turner Model 20E luminometer, over a 30minute period. T1/2 values were then calculated from the decay curves.The results of these determinations are given in Table IV below.

                  TABLE IV                                                        ______________________________________                                                    BACKGROUND AT HALF LIFE OF                                        DIOXETANE   0.4 mM (TLU)  ANION (min.)                                        ______________________________________                                        1. 0.05M Sodium Carbonate/Sodium Bicarbonate, 1 mM Magnesium                  Chloride, pH 9.5                                                              AMPPD       1.96          2.42                                                A-OH-AMPPD  1.20          1.33                                                B-OH-AMPPD  1.72          1.49                                                Cl-AMPPD    0.93          1.08                                                Br-AMPPD    1.35          0.99                                                2. 0.1M Diethanolamine, 1 mM Magnesium Chloride, 0.02% Sodium                 Azide, pH 10.0                                                                AMPPD       2.09          2.26                                                A-OH-AMPPD  0.95          1.05                                                B-OH-AMPPD  1.32          1.31                                                Cl-AMPPD    0.77          0.86                                                Br-AMPPD    1.13          0.56                                                ______________________________________                                    

EXAMPLE XIX

Alkaline phosphatase (calf intestine; Biozyme) was diluted 1-1,000,000to generate a stock solution, concentration 2.54×10⁻¹² M. A series oftubes was prepared, in duplicate, containing 450 μl of an aqueous 0.1Mdiethanolamine solution containing 1 mM magnesium chloride and 0.02%sodium azide, pH 10, plus 0.1% of one of the enhancer polymers specifiedbelow and 4.4×10⁻⁴ M of a 1,2-dioxetane: AMPPD or the correspondingchloroadamant-2'-ylidene compound.

Fifty μl of alkaline phosphatase stock solution was then added to givesamples containing the following enzyme concentrations:

2.54×10⁻¹² M

8.49×10⁻¹³ M

2.83×10⁻¹³ M

9.45×10⁻¹⁴ M

3.15×10⁻¹⁴ M

1.05×10⁻¹⁴ M

3.49×10⁻¹⁵ M

1.16×10⁻¹⁵ M

3.88×10⁻¹⁶ M

1.29×10⁻¹⁶ M

4.31×10⁻¹⁷ M

The final concentration of 1,2-dioxetane in each tube was 4×10⁻⁴ M; thefinal concentration of enhancer polymer was 0.09%. Five second integralswere recorded at 5 and 20 minutes following 1,2-dioxetane addition.

FIGS. 13 and 14 are dose response curves for alkaline phosphatasedilution with the chloroadamant-2'-ylidene 1,2-dioxetane plus BDMQ at 5and 20 minutes after dioxetane addition, as compared to the doseresponse curves for AMPPD plus BDMQ. FIGS. 15 and 16 show alkalinephosphatase dilutions detected with the chloroadamant-2'-ylidene1,2-dioxetane plus BDMQ-fluorescein ("emerald") andpoly(vinylbenzyltributylammonium chloride) ("TBQ")-fluorescein, at 5 and20 minutes after substrate addition, as compared to AMPPD plus the sameenhancer polymers.

EXAMPLE XX

pBR 322 plasmid (Biogen, Inc., Cambridge, Mass.) containing an insert ofTPA sequence was digested with MSP1 restriction enzyme. Chemicalcleavages were performed as described in Maxam, et al., PNAS, 74, 560(1977) to yield G, AG, AC, TC and C--and one other, T,--as described byRubin, et al., Nucleic Acids Research, 8, 4613 (1980). One seventh ofeach reaction tube's contents was loaded per lane onto 0.4 mmTBE-gradient sequencing gel (60 cm in length). After 4 hours ofelectrophoresis, DNA was electrotransferred to a BIODYNE A nylonmembrane (0.45 μm) and treated with ultraviolet light to fix the DNA tothe membrane's surface.

Next, the membrane was dried, prehybridized for 30 minutes at 45° C. inaqueous buffer solution containing 1% BSA, 0.5M sodium phosphate and 7%SDS, pH 7.2, then hybridized for 2 hours at 45° C. with 10 μl of NNBsnap direct alkaline phosphatase conjugated probe (Molecular Biosystems,Inc., San Diego, Calif.) in 40 ml of the above-described BSA buffersolution. The membrane was then washed twice in aqueous 5×SSC/1% SDS(for 5 minutes each time) at 45° C., twice in aqueous 1×SSC/1% SDS (for5 minutes each time) at 45° C., once in an aqueous solution containing125 mM sodium chloride, 50 mM Tris and 1% Triton X-100 detergent, pH8.0, twice in aqueous 1×SSC (for 1 minute each time) at roomtemperature, and finally twice (for one minute each time) at roomtemperature in an aqueous solution containing 0.1M diethanolamine, 1 mMmagnesium chloride and 0.02% sodium azide at pH 10.0.

The membrane was then wetted with aqueous AMPPD solution (0.25 mM),wrapped in Saran wrap, and exposed to Kodak XAR X-ray film for 40minutes. A five minute exposure was then taken 1 hour after AMPPDaddition. The sequence images are shown in FIG. 17(1).

Repeating this entire procedure using the correspondingchloroadamant-2'-ylidene 1,2-dioxetane compound gave the sequence imagesshown in FIG. 17(2).

The above discussion of this invention is directed primarily topreferred embodiments and practices thereof. It will be readily apparentto those skilled in the art that further changes and modifications inthe actual implementation of the concepts described herein can easily bemade without departing from the spirit and scope of the invention asdefined by the following claims.

We claim:
 1. An enol ether of the formula: ##STR22## wherein: X and X¹each represent, individually, hydrogen, a hydroxyl group, a halosubstituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkylgroup, a halo (lower) alkyl group, a phenyl group, a halophenyl group,an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amidegroup, with at least one of X and X¹ being other than hydrogen;Yrepresents a lower alkyl group; and Y' represents hydrogen, a loweralkyl group, a lower alkenyl group, an aralkyl group having up to 20carbon atoms, an alkali metal cation an acyl group having from two toabout 14 carbon atoms, inclusive, ##STR23## an alkali metal cation,where M represents, independently, a proton, an alkali metal cation,ammonium, substituted ammonium, quaternary ammonium or an H⁺ pyridiniumcation.
 2. A compound of claim 1 wherein X is hydroxyl and X¹ ishydrogen.
 3. A compound of claim 1 wherein X is chloro and X¹ ishydrogen.
 4. A compound of claim 1 wherein X is bromo and X¹ ishydrogen.
 5. A compound of any one of claims 2-4, inclusive, wherein Yis methyl.
 6. A compound of claim 5 wherein Y¹ is ##STR24##
 7. Acompound of claim 6 wherein each M is sodium.
 8. Disodium3-(methoxy-5-hydroxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenylphosphate.
 9. Disodium 3-(methoxy-5-chlorotricyclo 3.3.1.1³,7!dec-2-ylidenemethyl)phenyl phosphate.
 10. Disodium3-(methoxy-5-bromotricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)phenylphosphate.
 11. The enol ether of claim 1, wherein Y is a C₁ -C₂₀ alkylsubstituted with a perfluoralkyl group of 1-7 carbon atoms.
 12. The enolether of claim 11, wherein Y is a lower alkyl substituted with atrifluoromethyl group.
 13. The enol ether of claim 12, wherein Y is a C₁alkyl.
 14. A dioxetane of the formula ##STR25## wherein X and X¹ eachrepresent, individually, hydrogen, a hydroxyl group, a halo substituent,a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenylgroup, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group,a cyano group or an amide group, with at least one of X and X¹ beingother than hydrogen;wherein Y is a moiety selected from the groupconsisting of a phosphate, galactoside, acetate,1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, adenosinetriphosphate, adenosine diphosphate, adenosine monophosphate, adenosine,alpha-D-glucoside, beta-D-glucoside, alpha-D-mannoside,beta-D-mannoside, beta-D-fructofuranoside, beta-D-glucosiduronate,p-toluenesulfonyl-L-arginine ester, and p-toluenesulfonyl-1-arginineamide; wherein R is a C₁ -C₂₀ alkyl group; and wherein R¹ is aperfluoralkyl group of 1-7 carbon atoms.
 15. The dioxetane of claim 14,wherein R is C₁ alkyl.
 16. The dioxetane of claim 15, wherein R¹ istrifluoromethyl.